Process for the detection of oxidative stress and kit for its implementation

ABSTRACT

The present invention relates to a process for detecting oxidative stress in a sample and to a kit for this implementation. According to one embodiment the present invention provides a method for the detection of oxidative stress in an individual carrying a risk factor for oxidative stress comprising determining the risk factor for oxidative stress of said individual; selecting at least two oxidative stress markers being increased or decreased for said risk factor relative to healthy individuals; and measuring the amount of said at least two oxidative stress markers in a sample obtained from said individual.

This application is the U.S. National Phase of PCT/EP02/09079, filedAug. 13, 2002, which claims priority to Belgian Application No.2001/0545, filed Aug. 14, 2001.

The present invention relates to a process for detecting oxidativestress in a blood sample and to a kit for the implementation of thisprocess.

Definition of Oxidative Stress.

Some 98% of the oxygen taken up by our organism is reduced to water atthe level of the mitochondrial respiratory chain, in reactions catalyzedby cytochrome oxidase complexes. One molecule of dioxygen yields twomolecules of water, by direct capture of four electrons and fourprotons. Yet oxygen can also undergo stepwise reduction, electron byelectron. This leads to formation of highly toxic oxygen species, theactivated oxygen species (AOS), the first of which is the superoxideradical anion (O₂. ⁻). The chemical structure of the superoxide anionincludes an ‘unpaired’ electron (symbolized by a dot), an essentialfeature of free radicals. The presence of a free electron makes theradical highly unstable and very reactive as a powerful oxidant. Thesuperoxide anion can in turn capture an electron to become O²⁻ which, inthe presence of two protons, will form two new, highly oxidativeactivated oxygen species: hydrogen peroxide (H₂O₂) and single oxygen(¹O₂). These two molecules are not free radicals, since all of theelectrons in these chemical structures are paired. The electron capturedby the superoxide anion can come from another superoxide anion, thisleading to spontaneous dis-mutation of the superoxide anion. Coexistenceof the superoxide anion with, hydrogen peroxide can yield yet anotherhighly reactive activated oxygen species: the hydroxyl radical OH. Thisreaction requires the presence of a transition metal such as iron orcopper, acting as a catalyst. On the free-radical scene, these metalsare key players that cannot be ignored.

By forming AOS, oxygen can aggressively compromise cell integrity. Theabove-mentioned oxygen species, and particularly free oxygen radicals,have extremely short life spans; they interact with a wide variety ofbiological substrates such as nucleic acids, nucleotides, proteins,membrane lipids, and lipoproteins. AOS can produce breaks indeoxyribonucleic acid (DNA) and thus alter the genetic message. In thecytoplasm, AOS transform molecules such as NADH or NADPH and thus alterthe redox status of the cell and the activity of enzymes using thesesubstrates. The action of AOS markedly modifies the primary, secondary,and tertiary structure of proteins, to the point of denaturing them andforming insoluble aggregates (cell debris). Depolymerization of proteinssuch as collagen and elastin is a good example of the deleterious actionof AOS. The protease inhibitor α-1-antitrypsin (which inhibits elastaseand trypsin) is rapidly inactivated by free oxygen radicals. When redblood cells are in contact with AOS for just a few minutes, theirhemoglobin is altered; iron is released from the heme, and hemolysis ofthese cells is increased. Membrane phospholipids are essentialconstituents of cell architecture. They contain polyunsaturated fattyacids (PUFA), favored targets of free oxygen radicals. The hydrogenlocated between the double bonds of PUFA is readily pulled off by thehydroxyl radical (OH.), which thus converts the fatty acid (RH) to afree radical (R.). In the presence of oxygen and iron, lipidperoxidation is induced, propagating from fatty acid to fatty acid viaformation of free lipid alkoxyl radicals (RO.), peroxyl radicals (ROO.),and lipoperoxides (ROOH). The result is a major alteration of membranefluidity, possibly leading to cell death. Rich in PUFA, lipoproteins areparticularly sensitive to the action of AOS. Oxidized lipoproteins nolonger correctly transport cholesterol. In addition, they are recognizedby blood macrophages and accumulate inside them. The macrophages thentake on the appearance of foam cells, which attach to artery walls. Thisis the mechanism by which oxidized lipoproteins contribute to increasingthe risk of cardiovascular disease.

Recent studies have shown that AOS can also play a role at the molecularlevel. An example is their action on NF-κB, a B-lymphocyte-specifictranscription factor. Maintained inactive in the cytoplasm, NF-κB can beinduced in a wide variety of cell types by various factors, includingcytokines, infectious agents, and also AOS acting as second messengers.Thioredoxin (TRX), a protein induced by oxidative stress, also increasesthe activity of NF-κB by modifying the redox regulation of glutathione(GSH). Once activated, NF-κB migrates to the nucleus of the cell, whereit can transactivate target genes. It is thus involved in the synthesisof many mediators of the immune and inflammatory responses (cytokines,complement). Several viruses such as HIV also depend on NF-κB toreplicate in the cell.

Our organism is always producing AOS but, as will appear in the nextsection, this production is perfectly regulated by protective systems.AOS thus play an important physiological role, as illustrated in thefollowing examples. Phagocyte cells, neutrophils, eosindphils, andmonocytes/macrophages ensure the phagocytosis and destruction of foreignmicro-organisms. The cytotoxicity of these cells is due to their abilityto shift from a quiescent form to a highly activated form characterizedby production of abundant intracellular AOS capable of attacking themembrane of the phagocytosed micro-organism.

Recent studies have shown that in the fertilization process, aspermatozoa secretes large quantities of AOS so as to pierce themembrane wall of the ovum. Under physiological conditions, endothelialcells release a substance called EDRF (endothelium derived relaxingfactor), which plays an important role in regulating vascular tonus,because it has muscle-relaxing properties. It has been clearlydemonstrated that this EDRF acts like the free nitrogen monoxide NO.radical.

Source of in Vivo AOS Production.

AOS overproduction is attributable to several biochemical mechanisms:

Certainly the most important mechanism is activation of white bloodcells by various external stimuli such as endotoxins, interleukins, orcomplement fragments present in excess in the organism in variouspathological situations. The AOS produced via this activation are thensecreted into the extracellular medium and can thus attack healthyorgans and tissues and trigger acute inflammation. White blood cellsalso release an enzyme called myeloperoxidase (MPO). In the presence ofhydrogen peroxide, this enzyme catalyses oxidation of the chloride ionto a powerful oxidant, hypochloric acid (HOCl), better known as“chlorine bleach”.

We have seen that under physiological conditions, mitochondria transformabout 2% of their oxygen into AOS. Yet animal studies have shown thatthis percentage increases during aging, as a result of gradualderegulation of electron transport in the respiratory chain.

Deregulation of electron transport in the mitochondria is also observedin all ischaemia-reperfusion processes. Ischaemia, i.e. oxygendeprivation, in a tissue causes cell lesions in proportion to itsduration. To preserve tissue viability, reperfusion must take placewithin a certain time limit, which depends on the tissue considered.Several experimental and clinical studies have shown, however, thatmajor cell damage occurs when the blood flow-is restored, as a result ofexplosive production of AOS in the minutes following reperfusion. Theconversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO)(these two enzymes act in the last stages of purine catabolism) is alsoa major source of AOS. Clinically, ischaemia-reperfusion is an importantaspect of stroke and organ transplantation. In cases of oxidativestress, endothelial cells abundantly produce both the superoxide anionand the nitrogen monoxide radical (NO.). Interaction of these radicalshas two effects: production of highly toxic peroxynitrites (HOONO) andvasoconstriction due to loss of the vasodilating properties of NO.

AOS, and especially the superoxide anion, can ‘activate’ iron byreleasing it from the storage protein ferritin; the released iron canthen initiate free-radical reactions.

Hemoglobin can become a potential oxidant via direct formation of AOS orvia. release of heme iron in free form.

In blood platelets, the conversion of arachidonic acid to prostaglandinsor leukotrienes, catalyzed respectively by cyclo-oxygenase andlipo-oxygenase, is a potential source of AOS and lipoperoxideproduction.

Antioxidant Defences.

To protect itself against the potentially harmful effects of oxygen, ourorganism has developed two types of defense systems.

A primary defense system composed of antioxidant enzymes and compounds.The function of these factors is to prevent the initiation orpropagation of free-radical reactions. Enzymatic protection is ensuredby superoxide dismutatse (SOD), which destroys the superoxide anion,catalase, which eliminates hydrogen peroxide, glutathione peroxidase, aselenium-dependent enzyme, which converts lipid peroxides to harmlessproducts, iron-chelating proteins (ferritin, transferritin), whichmaintain in an inactive state iron that would otherwise be capable ofcatalyzing free radical reactions. In addition, free radicals aredestroyed by scavengers, which react directly with the radicals to formoxidized derivatives. For example, glutathione (GSH) reacts with the OH.radical to form oxidized glutathione (GSSG), by a mechanism involvingformation of a GS. (thiyl) radical, followed by combination of two suchradicals. Other examples of scavengers include uric acid, bilirubin,glucose, thiol proteins (SH proteins), and a group of four vitamins:vitamin A (β-carotene), C (ascorbic acid), E (α-tocopherol), and Q(coenzyme Q-10 or ubiquinone). Remarkably, these antioxidants can actsynergistically against free radicals. An example is the cooperationbetween glutathione and vitamins C and E. When it reacts with a lipidradical R., the lipid is regenerated and vitamin E becomes a tocopherylradical. Vitamin E is regenerated from this radical through the actionof vitamin C, which in turn becomes a radical. This radical then reactswith reduced glutathione, to form vitamin C and a thiyl radical. Two ofthe latter combine to yield GSSG.

A secondary defense system is composed of proteolytic enzymes. The roleof this system is to prevent accumulation of oxidized proteins and DNAin the cell and to degrade their toxic fragments. Phosholipases, DNAendonucleases and ligases, and macroxyproteinases are among the mainenzymes forming this last line of defense against AOS.

Pathologies Associated with Increased Oxidative Stress.

Some pathological situations lead to overproduction of AOS. In suchsituations, an organism's natural defense systems are rapidlyoverpowered. Generally speaking, oxidative stress is defined as a majorimbalance between anti- and pro-oxidants, in favor of the latter,leading to cell damage that is often irreversible.

Overproduction of AOS is considered responsible for about a hundreddifferent pathologies. These fall into 6 major categories: effects dueto chemical toxicity (ozone, paraquat) or xenobiotic toxicity, effectsdue to radiation (γ rays), the hyperoxygenation syndrome (retinopathy ofthe newborn), inflammatory conditions (rheumatoid arthritis), effects ofischaemia-reperfusion (organ transplantation), degenerative conditions(aging, cancer). Many situations/pathologies are associated with AOSoverproduction:

-   -   Kidneys: transplantation, glomerular nephritis    -   Lungs: respiratory distress syndrome, asthma    -   Heart: coronary thrombosis, transplantation    -   Skin: burns, sunlight exposure, psoriasis, dermatosis    -   Brain: trauma, Parkinson's disease, neurotoxins, dementia    -   Joints: rheumatoid arthritis    -   Gastrointestinal tract: diabetes, pancreatitis, endotoxemia,        intestinal ischaemia    -   Eyes: cataract, retinopathy (newborn), retinal degeneration    -   Blood vessels: atherosclerosis    -   Red blood cells: Fanconi's anemia, malaria    -   Multiple organs: inflammation, ischaemia-reperfusion, drug        toxicity, iron overload, nutritional deficiency (Se), alcohol,        radiation, cancer, aging, AIDS.    -   Liver: Hepatitis C

Excessive exercise can also create an oxidative stress in athletes,associated with muscle fatigue and injuries.

When produced in small amounts, AOS trigger an adaptive cell responsecharacterized by increased cell growth, overexpression of antioxidantenzymes (superoxide dismutase), and expression of genes coding for manyproteins. Cells exposed to low doses (5 μmoles) of hydrogen peroxide,for example, thus become very resistant to higher concentrations of thisoxidant. When produced in excess, AOS attack all the above-mentionedsubstrates and thus cause often-irreversible cell damage and subsequenttissue necrosis. Between these two extreme situations, there is onewhere oxidative stress leads to apoptosis, i.e. programmed cell death.This phenomenon involves massive proteolysis and a change in genetranscription, leading to chromatin condensation and DNA fragmentationin the endosomes. Cell integrity is preserved, unlike what happens inpathological necrotic cell death. Before birth, more than one cell outof six dies by apoptosis. Apoptosis is also characterized by majorleakage of GSH, rendering the cell more sensitive to oxidative stress.

Antioxidant Therapy.

When oxidative stress largely overwhelms the natural defense systems,recourse to antioxidant therapy is necessary so as to limit the harmfulaction of AOS. In addition to the natural antioxidants described above,there exist exogenous antioxidants, both natural and synthetic.

Each antioxidant has its specificity. Different ones act at differentsteps in the AOS production chain. Four major categories can bedistinguished:

-   -   1. molecules interacting with AOS, either enzymatically (SOD,        catalase, glutathione peroxidase) or directly        (dimethylsulfoxide, vitamins A, C, and E, ubiquinone,        21-aminosteroids, probucol, captopril, propofol, lipoic acid,        flavonoids or P vitamins, . . . );    -   2. molecules reinforcing the antioxidant defenses of cells.        Examples include N-acetylcysteine (NAC), which increases the        intracellular GSH concentration, and certain metals acting as        cofactors. Copper and/or zinc administration reinforces SOD        activity in red blood cells, while selenium reinforces        glutathione peroxidase activity in these cells;    -   3. iron-chelating molecules, one of the best examples being        desferioxamine. This compound is notably used to treat        idiopathic hemochromatosis. Both the 21-aminosteroids and the        flavonoids present in large amounts in Gingko biloba possess        this interesting iron-chelating capacity;    -   4. inhibitors of enzymes responsible for free radical formation.        The best known is allopurinol, a xanthine oxidase inhibitor used        regularly in surgery to limit the effects of        ischaemia-reperfusion. Also worth mentioning are certain        anti-inflammatory agents (sulfasalasine, 5-aminosalicylic acid)        and other products such as ceftazidine and pentoxyfylline, which        regulate AOS production by activated white blood cells.

Individuals do not possess identical anti-oxidant potential, as suchpotential is a function of one's eating habits, lifestyle, geneticcharacteristics or the environment where one lives. At present,determining an individual's status of oxidafive stress (SSO) has becomea priority subject in terms of disease prevention, since numerousstudies clearly display a strong link between the alteration of theanti-oxidant defense system and the increased effect of thecardiovascular diseases and cancers.

At the moment, using the development of sensitive and specifictechniques which may be used in routine, (on a routine basis 20 assayscovering the measure of the anti-oxidants of the trace elements,indicators of stress of the metabolism of iron and of the markers ofoxidative stress at the level of the lipids, of the proteins and of theDNA), it is possible to establish a complete blood check-up of the SSOof an individual.

Evidence of in Vivo Oxidative Stress: Classical Assays.

In serum or plasma, the oxidative stress status (SSO) will be estimatedin four ways: (i) by measuring antioxidant levels, (ii) by determiningtrace element levels, (iii) by analyzing the status of iron in theblood, and (iv) by detecting oxidative damage to various biologicalsubstrates.

A. Antioxidants.

Vitamin A: Certain carotenoids act as vitamin A precursors, yielding thevitamin through degradation. Vitamin A plays an essential role in visualperception and prevents oxidation of several biological substrates, e.g.the polyunsaturated fatty acids of cell membranes.

Vitamin C: This vitamin reacts with free hydrophilic oxygen-containingradicals. Its plasma level drops very rapidly during oxidative stress.Vitamin C is also involved in regenerating vitamin E. Because vitamin Cis very labile, it cannot be assayed correctly unless precautions aretaken to preserve the sample (stabilization of the plasma withmetaphosphoric acid).

Vitamin E: This term encompasses a group of compounds called tocopherols(α, β, γ, δ). The most biologically active isomer is α-tocopherol,assayed in this study. It reacts with lipid radicals and thus preventspropagation of lipid peroxidation in cell membranes. It acts in closesynergy with vitamin C. Its plasma concentration must be standardizedwith respect to cholesterol.

Reduced glutathione (GSH)/oxidized glutathione (GSSG): The tripeptideglutathione acts at several levels against oxidative stress. It caninteract directly with activated oxygen species (AOS), but it is mainlyused as a substrate of glutathione peroxidase, which eliminatesperoxidised lipids. The GSH/GSSG ratio is one of the most sensitiveindicators of oxidative stress in an individual.

Protein thiols (PSH): Most proteins have thiol groups (—SH), which reactvery-readily with AOS.

Glutathione peroxidase (GPx): Located in red blood cells, GPx requiresselenium and reduced glutathione for its antioxidant activity. Its roles to eliminate hydrogen peroxide and lipid peroxides formed when AOSreact with polyunsaturated fatty acids.

Superoxide dismutase (SOD): These are metalloproteins of which twovarieties are distinguished in man, according to the metal(s) present atthe active site. One type contains copper and zinc, while the otherrequires manganese to be active. The role of these enzymes is toregulate production of the superoxide anion, the first toxic species tobe formed from oxygen.

Total plasma antioxidant capacity (analysis of hydrophilic andlipophilic antioxidants): This test evaluates the capacity of plasma toinhibit the production of AOS generated by an in vitro system (the TRAPmethod). It is thus a screening method that estimates the sum of allindividual antioxidant activities present in various biologicalenvironments.

B. Trace Elements.

Selenium, Copper, Zinc: These three oligoelements are indispensable tothe function of antioxidant enzymes (selenium for GPx, copper and zincfor SOD). Zinc intake leads in the long term to induction ofantioxidants such as metallothionines. Zinc also protects the thiolgroups of proteins. Zinc can partially inhibit reactions in which copperor iron induces the formation of activated oxygen species. For thisreason, the ratio of copper to zinc in the blood may provide interestinginformation on an individual's level of oxidative stress.

C. Indicators of Oxidative Stress.

Lipid peroxides—oxidized LDL—antibodies against oxidized LDL:Polyunsaturated fatty acids (essential components of cell membranes andlipoproteins) are a preferential target of AOS. These three tests areused to evidence lipid peroxidation. Many studies show a link between,on the one hand, increased LDL oxidation and an increased titre ofantibodies against oxidized LDL, and, on the other hand, the appearanceof cardiovascular disease, progression of atherosclerosis, and thepresence of diabetes.

8-hydroxy-2′-deoxyguanosine (8-OH-dG): AOS react with high affinity withsome of the bases that constitute DNA. Guanine is readily transformed to8-hydroxy-2′-deoxyguanosine (8-OH-dG), which is normally eliminated byDNA repair enzymes. If these systems are deficient, 8-OH-dG accumulatesin the DNA, causing mutations implicated in cancer formation. Whenpresent in 24-hour urine samples, this marker must be standardized withrespect to creatinine.

Carbonyl assay. Oxidative modifications of intracellular proteins havebeen suggested to play a key role in the causation ofsenescence-associated losses in physiological functions because oxidizedproteins often lose catalytic function and undergo selectivedegradation. Addition of carbonyl-containing adduct to the side chainsof amino-acid residues (lysine, arginine, . . . ) is arguably the mostwell characterized, age-associated, post-translation structuralalteration in proteins.

Myeloperoxidase: When activated, neutrophils increase their oxygenconsumption by 400%. This leads to massive production of AOS and to therelease of proteolyic enzymes (elastase) and myeloperoxidase (MPO). MPOis involved in the development of oxidative stress, being responsiblefor the formation of hypochlorous acid, a powerful oxidant. An increasedplasma MPO level is thus a specific indicator of neutrophil activation,and this an indirect indicator of the presence of AOS, occurring in allinflammatory processes.

Glucose: By auto-oxidation, glucose yields AOS and glyoxal in largequantities. Glyoxal binds to the amine groups of proteins and henceleads to the appearance of “old proteins” with carboxymethyl-lysineresidues. These have the capacity to bind copper and to induce lipidperoxidation, which in turn increases glyoxal production. Glucose itselfcan bind to hemoglobin to produce glycated hemoglobin. These markersincrease, of course, in patients with diabetes, considered a situationof major oxidative stress. Yet aging can also cause these markers toaccumulate.

D. Iron Metabolism.

Proteins that bind metal ions: When iron or copper is in a free form, itcan catalyze lipid peroxidation and the production of AOS. The capacityof some proteins to bind iron (transferrin, ferritin) or to transportcopper (ceruleoplasmin) is an essential preventive antioxidant factor.In the absence of sample hemolysis, an increased saturation oftransferritin in iron is an indirect indicator of oxidative stress.

E. Miscellaneous.

Elevated blood levels of homocysteine (a sulfur-containing amino acid)have been linked to increased risk of premature coronary artery disease,stroke, and thromboembolism (venous blood clots), even among people whohave normal cholesterol levels. Abnormal homocysteine levels appear tocontribute to atherosclerosis in at least three ways: (1) a direct toxiceffect that damages the cells lining the inside of the arteries, (2)interference with clotting factors, and (3) oxidation of low-densitylipoproteins (LDL).

Detecting an in vivo oxidative stress is thus a long, complex andexpensive process which limits its application to medicine, althoughways to treat an oxidative stress exist.

Moreover, these diagnostic tests do give a little information on what isgoing on at the level of the cells, especially at the level of thelymphocytes which metabolism is very dependent on the EOA. Therefore, atthe moment no test exists that allows information at this level to beobtained.

One objective of the present invention is to provide a diagnostic testmethod that allows the status of oxidative stress (SSO) (preferably atthe cellular level) to be evaluated quickly and easily.

Furthermore, the invention seeks to provide a kit suitable forperforming said method. Further technical problems will be apparent fromthe subject matter of the claims, the disclosure of which isincorporated hereby.

According to the present invention, the term “risk factor” means anypossible physiological and/or pathological condition wherein anindividual might be affected by oxidative stress. In particular, intensephysical exercise, effects due to chemical toxicity or xenobiotictoxicity, effects due to radiation, hyperoxyegenation syndrome,inflammatory conditions, effects of Ischaemia-reperfusion anddegenerative conditions are considered. Below, physiological and/orpathological conditions being characterized by a risk factor foroxidative stress are exemplified.

According to the present invention, the term “anti-oxidant” means anysubstance capable of protecting from the potentially harmful effects ofoxygen; in particular, capable of preventing the initiation offree-radical reactions.

According to the present invention, the term “pro-oxidant” means anysubstance capable of directly or indirectly supporting the initiation ofpropagation of free-radical reactions.

According to the present invention, the term “oxidative stress marker”means any marker for oxidative stress known in the art or as describedherein.

According to the present invention, the term “protein implied inapoptosis” means any protein being capable of promoting apoptosis, i.e.,programmed cell death, or any protein or RNA that is involved in, orregulates, the apoptosis pathway. In particular, molecules beingassociated with CD95/CD95 ligand (Fas/Fas ligand) are envisaged.

According to the present invention, the term “DNA chip” should beunderstood as synonymous with microarray or biochip, not being limitedto a particular length of nucleic acids or oligonucleotides attachedthereto.

According to the present invention, the term “enzyme” also encompassespolypeptides not necessarily exhibiting catalytic activity.

According to the present invention, the term “transcription factors”means any DNA or RNA binding protein or RNA regulating genetranscription and translation, or any RNA, protein, or protein cascaderegulating them.

According to the present invention, the term “DNA repair enzyme” meansany protein or RNA recognizing DNA modifications or any RNA, protein, orprotein cascade regulating them.

According to the present invention, the term “stress protein” means anyprotein or RNA, expression of which is modified under a cellular stressinduced by physical, chemical, or biological conditions outside theirnormal physiological values.

Surprisingly, the present inventors have discovered that the oxidativestress measured in individuals exposed to different degrees andqualities of oxidative stress differs in its profile of oxidative stressmarkers, i.e. in the amount of the different oxidative stress markers,when a large variety of stress markers is assessed. For example, theprofiles of oxidative stress markers have been obtained from healthyindividuals, cardiac patients, hemodialysis patients, people havingheavy physical exercise (such as half-marathon athletes and top soccerplayers.) Although it would have been expected that the amount ofoxidative stress markers is increased in the same manner in patients andathletes showing exhaustion due to physical exercise, it has turned outthat different oxidative stress markers are selectively induced uponexposure to different sources of oxidative stress; cf. table III. Asapparent from tables I and III, some oxidative stress markers areincreased under a certain physiological and/or pathological condition,whereas under the same condition different oxidative stress markers maybe decreased see e.g. the value for hemodialysis patients from TableIII, wherein the average values for lipid peroxide and oxidized LDL areincreased, whereas the average values for superoxide dismutase (SOD) andselenium are decreased. These findings have important impacts on thediagnosis and possible therapy of diseases based on oxidative stress.

The present invention allows for the first time a significant reductionin the number of oxidative stress markers to be tested, because onlyseveral oxidative stress markers coming from the literature haveactually been found to be increased or decreased in individuals having aparticular physiological and/or pathological condition when compared tohealthy individuals. Additionally, the selection of oxidative stressmarkers that have been observed to be increased or decreased in aparticular physiological and/or pathological condition is reducing thenumber of false negatives of previous oxidative stress tests that eitherused markers which are, under the particular physiological and/orpathological condition, neither been increased nor decreased, orincluded the markers that have been observed to be increased ordecreased in a particular physiological and/or pathological conditioninto a larger panel of non-varying markers, computing an average valuethat is staying within its normal range. The present invention allowsfor the risk factor-specific testing of individuals which enables afine-tuning of diagnosis and possibly also therapy of the underlyingoxidative stress syndrome in the physiological and/or pathologicalcondition. Further, the present invention allows for the first time arisk factor-specific evaluation of the data obtained from testing theoxidative stress markers selected according to the present inventionwhich also facilitates the physician to choose a patient-specifictreatment regimen.

In one embodiment, the present invention provides a method fordetermining oxidative stress markers in a group of individualscomprising the steps of:

-   -   a) Determining the risk factor for oxidative stress in said        group;    -   b) Measuring the amount of at least 10 different oxidative        stress markers in a sample obtained from each of said group of        individuals; and    -   c) Comparing the amount of each of said oxidative stress markers        with the amount of each of said oxidative stress markers        measured in a group of healthy individuals, thereby determining        the oxidative stress markers being increased or decreased in        said group of individuals carrying a risk factor for oxidative        stress relative to healthy individuals.

The individual may be a human or animal.

The risk factors for oxidative stress are determined by the physicianaccording to general anamnesis. Exemplary risk factors are outlinedbelow.

The measurement of the amount of the oxidative stress markers isdetermined as outlined below.

Preferably, for statistical reasons, the group comprises at least 10individuals, preferably more than 50. Subsequent to measurement of theamount of the different oxidative stress markers, the average value isobtained and used for the subsequent comparison step. Also for the groupof healthy individuals, at least 10 healthy individuals should be takenin order to obtain reliable average values. Preferably, the groupconsists of only one gender, (i.e. either male or female), sincevariations in the amount of oxidative stress markers among men and womenhave been observed; cf. FIG. 1.

In a further embodiment, the present invention provides a method for thedetection of oxidative stress in an individual comprising:

-   -   a) Determining the risk factor of said individual;    -   b) Selecting at least 2 oxidative stress markers being increased        or decreased for said risk factor relative to healthy        individuals;    -   c) Measuring the amount of at least 2 of said oxidative markers        in a sample obtained from said individual.

Preferably, the oxidative stress markers being increased or decreasedfor said risk factor are determined by the above method according to thepresent invention. The sample is preferably a blood sample beingobtained by or under the control of a physician.

In a preferred embodiment, said method for the detection of oxidativestress in an individual further comprises the step of evaluating theresult in the context of said risk factor without restricting orlimiting the physician in any way. It is suggested that the results fora particular oxidative stress marker within a certain individualcarrying a risk factor for oxidative stress should be seen in relationto the average value for said oxidative stress marker of the group ofindividuals carrying the same risk factor for oxidative stress, therebypossibly allowing an estimation of the degree of oxidative stress insaid individual carrying the particular risk factor.

The risk factor mentioned in the above methods may be selected fromunbalanced diet, smoking habits, exposure to toxic environment, medicalsurgery, intense physical exercise, and diseases affecting the kidneys,lungs, heart, skin, brain, joints, gastrointestinal tract, eyes, bloodvessels, red blood cells, liver and multiple organs.

Preferably, the diseases are selected from transplantation, glomerularnephritis, respiratory distress syndrome, asthma, coronary thrombosis,burns, sunlight exposure, psoriasis, dermatosis, trauma, Parkinson'sdisease, neurotoxins, dementia, rheumatoid arthritis, diabetes,pancreatitis, endotoxemia, intestinal eschemia, cataracts, retinopathy,retinal degeneration, arteriosclerosis, Fanconi's anemia, malaria,inflammation, ischaemia-reperfusion, drug toxicity, iron overload,nutritional deficiency, alcohol, radiation, cancer, aging, HCV infectionand AIDS.

It should be noted that the above list of diseases is non-limited withrespect to indications for using the methods according to the presentinvention. Apart from the above diseases, any physiological and/orpathological situation leading to overproduction of AOS is envisaged bythe present invention.

The oxidative stress markers that are useful in the practice of themethods according to the present invention may be selected from thegroup consisting of antioxidants, trace elements, indicators ofoxidative stress, iron metabolism markers, homocysteine, enzymes havingantioxidant functions, enzymes having pro-oxidant functions, enzymes forDNA repair, enzymes of the glutathione metabolism, stress proteins,proteins implied in apoptosis, transcription factors, cytokines andchemokines.

Preferably, the antioxidant is selected from vitamin A, vitamin C,vitamin E, reduced glutathione (GSH)Ioxidized glutathione (GSSG),protein thiols, glutathione peroxidase or superoxide dismutase.

Preferably, the trace element is selected from selenium, copper andzinc.

In a preferred embodiment, the indicator of oxidative stress is selectedfrom antibodies against oxidized LDL (low-density lipoproteins),8-hydroxy-2′-deoxyguanosine, myeloperoxidase, glucose, glyoxal, andoxidized proteins.

Preferably, the iron metabolism marker is selected from transferrin,ferritin, ceruloplasmin.

Preferably, the enzymes having antioxidant function may be selected fromcatalase, Mn-containing superoxide dismutase (SOD), copper and zinccontaining SOD, thioredoxine-1, thioredoxine reductase-1,peroxiredoxin-1, metallothioneine-1, L-ferritine and transferrinereceptor, antioxidant protein 2, ceruloplasmin, lactoferrin,selenoprotein P, selenoprotein W, frataxin, serumparaoxonase/arylesterase 1, serum paraoxonase/arylesterase 2, or serumparaoxonase/arylesterase 3.

Preferably, the enzymes having pro-oxidant function may be selected fromcyclooxygenase-2,5-lipoxygenase, c-phospholipase A2, phospholipase Aalpha, phospholipase D-1, myeloperoxidase, nitric oxide synthetase, Creactive protein, elastase, haptoglobin, NADH-cytochrome b5 reductase ordiaphorase A1.

Preferably, the enzyme for DNA repair may be selected from 8-oxoguanineDNA glycosylase.

Preferably, the glutathatione metabolism enzyme may be selected fromglutathione peroxidase, non-Se glutathione phospholipid hydroperoxide,phospholipid, gamma-glutamyl cysteine synthetase, and glucose6-phosphate dehydrogenase, extracellular glutathione peroxidase,glutathione peroxidase, glutathione peroxidase 2, glutathione peroxidase4, glutathione reductase, glutathione S-transferase, glutathionesynthetase, peroxiredoxin 1, peroxiredoxin 2, peroxiredoxin 3,peroxiredoxin 5, or thioredoxin 2.

Preferably, the stress protein may be an HSP protein, heme-oxygenase-1,heme-oxygenase-2, 150 kDa oxygen-regulated protein ORP150, 27 kDa HSP27,HSP90A, HSP17, HSP40 or HSP110.

Preferably, the protein implied in apoptosis may be FasL, CD95, tumornecrosis factor 1, Bcl-2, GADD153, GADD45, RAD50, RAD51B, RAD52, RAD54,p53 or Fas ligand.

Preferably, the transcription factor is selected from NFκB-α, c-Fos,C-jun, IκB-α, monoamine oxidase A, monoamine oxidase B or peroxisomeproliferative-activated receptor alpha.

Preferably, the cytokine or chemokine is selected from IL-1, IL-6, IL-8,IL-1 beta, IL-2 or TNF1 receptor associated protein.

The investigations performed by the present inventors have revealed thatparticular oxidative stress markers are increased or decreased inindividuals carrying certain risk factors.

According to a preferred embodiment, the oxidative stress markers beingincreased or decreased when the risk factor hemodialysis is assessed arecatalase, glucose 6 phosphate dehydrogenase, HSP70, 5-lipoxygenase,vitamin C, glutathione peroxidase, SOD, Se, lipid peroxide, oxidized LDLand homocysteine.

The increase or decrease may be either on the transcriptional leveland/or on the level of the expressed protein.

According to a further preferred embodiment, the oxidative stressmarkers being increased or decreased when the risk factor is cardiacsurgery are superoxide dismutase containing manganese, c-phospholipaseA2, H-ferritin, IL-8, nitric oxide synthase 2 (NOS2), vitamin C, vitaminE/cholesterol, glutathione peroxidase (GPx), antibodies against LDL andhomocysteine.

According to a further preferred embodiment, the oxidative stressmarkers being increased or decreased when the risk factor due tophysical exercise is assessed are HSP 70, NFκB-α, vitamin C, copper/zincratio, vitamin E/cholesterol, GPx and seric ion.

In athletes carrying the risk factor of heavy physical exercise but alsoexposed to muscle fatigue and injuries, it has also been observed thatthe oxidative stress markers being increased or decreased are vitamin E,vitamin E/cholesterol, GSH, GSH/GSSG ratio, zinc, GPx, GSSG, copper/zincratio, antibodies against oxidized LDL and oxidized proteins.

According to a further preferred embodiment, the oxidative stressmarkers being increased or decreased when the risk factor is representedby smoking are vitamin C, Se, GPx, antibodies against oxidized LDL andhomocysteine.

According to another preferred embodiment, the oxidative stress markersbeing increased or decreased when the risk factor is due to a dietlacking fruits are vitamin C, protein thiols, Se, GPx and homocysteine.

According to the present invention when the method for the detection ofoxidative stress in an individual is determined as described above,preferably not more than 22 different oxidative stress markers areselected for measuring the amount of the oxidative stress markers in asample obtained from said individual. Preferably not more than 15, morepreferably not more than 10, and particularly preferred not more than 5different oxidative stress markers being increased or decreased in thegroup of individuals carrying the particular risk factor of saidindividuals. As outlined above, the present inventors have recognizedthat not every single one of the oxidative stress markers known in theart are increased under conditions of oxidative stress in individuals,but that only some depending on the physiological and/or pathologicalsituation, i.e. the risk factor of the individuals are increased.Surprisingly, it has been found that several of the known oxidativestress markers are even decreased in said individuals, whereas oxidativestress markers different than those may be unchanged compared to healthyindividuals.

In the methods according to the present invention, the amount of theoxidative stress marker may either be determined by measuring theconcentration of the oxidative stress marker or by measuring theconcentration of the gene transcript/mRNA or corresponding cDNA encodingthe oxidative stress markers.

When the concentration of the oxidative stress marker is determineddirectly, then any method known in the art for the determination of saidoxidative stress marker is encompassed. The determination method mayencompass enzymatic, immunochemical or spectroscopic methods. Thepresent invention envisages direct or indirect determination methodssuch as, for example, radioimmuno assays.

When the amount of the oxidative stress marker is measured bydetermining the concentration of the gene transcript/mRNA encoding theoxidative stress marker, any method for quantitative determination ofgene transcripts known in the art is encompassed hereby. Forquantitative determination, it is preferred that the mRNA be convertedto the corresponding cDNA by the reverse transcriptase reaction. Todetermine the corresponding PCR product, quantitative orsemi-quantitative PCR methods may be applied. The determination methodmay also involve hybridization to genes or fragments or derivativesthereof attached to a solid matrix, such as a Northern Blot, forexample. The present invention also envisages the use of spectroscopicmethods.

It is particularly preferred that the amount of at least 2 of theoxidative stress markers is determined in parallel. The parallelperformance of the determination offers the advantage of high-speeddetermination being recommended in clinical practice.

In regards to this embodiment, it is preferred that the paralleldetermination of the oxidative stress markers is performed by using aDNA chip (microarray or biochip) as known e.g. van Berkum and Holstege,Biotechniques 2000, 29: 548-560. The chip used for the determination mayhave any configuration, it is preferred that it allows for duplicate,triplicate, or quadruplicate determinations.

According to another embodiment of the invention, a test kit is providedthat is suitable for performing the method for the detection ofoxidative stress in an individual. The test kit suitable for performingthe method according to the present invention comprises reagents capableof determining the amount of at least 2 oxidative stress markers.Preferably, the reagent capable of determining the amount of saidoxidative stress marker is specific to said oxidative stress marker.

According to a preferred embodiment, the reagent capable of determiningthe amount of said oxidative stress marker is an antibody. The antibodymay be monoclonal or polyclonal. Monoclonal antibodies may be obtainedby immunizing laboratory animals, removing the splenocytes, and fusingthem with tumor cells to obtain oxidative stress marker-specificantibodies by selection according to methods known in the art.Monoclonal antibodies, antibody fragments, or binding molecules may alsobe selected by using molecular display techniques such as phage display.

The reagent capable of determining the amount of said oxidative stressmarker may, in the event that the oxidative stress marker is determinedby the concentration of the gene transcript/mRNA or its correspondingcDNA by a probe being essentially complementary to said genetranscript/mRNA or a fragment thereof. It is preferred that the probecomprise at least 10 nucleotides being complementary to the genetranscript/mRNA or fragment thereof. To allow for detection, the probemay also comprise a label being suitable for detection. The label may beradioactive or non-radioactive.

Accordingly, the test kit may comprise an antibody and/or a probespecific to said oxidative stress marker.

According to a preferred embodiment, a process of detecting oxidativestress in a blood sample is provided comprising:

-   -   extracting of mRNA from cells of the blood sample,    -   reverse transcribing of said mRNA into cDNA, with labeling of        these cDNA compounds,    -   bringing these cDNAs into contact with a population of several        synthetic DNA fragments, selected in a way to realize a        molecular hybridization between the cDNAs and said synthetic DNA        fragments, in case of expression of oxidative stress in the        cells, and simultaneous detection of signals of said        hybridization, which correspond to some expressed genes.

According to a preferred embodiment, the mRNA is extractred frompurified lymphocytes.

This process is advantageous for evaluating in one step, by thegenetics, the SSO of a patient.

Evidence of In Vivo Oxidative Stress: Innovative DNA MicroarrayTechnology.

An embodiment of this invention is to detect genes expressed duringcellular oxidative stress with the plurality of said synthetic DNAsarrayed on a solid support (DNA-arrays). Several solid supports havebeen described, and methods to prepare and to use the arrays are known(Cheung et al. Nature Genetics Supplement 1999, 21:15-19; Bowtell,Nature Genetics Supplement 1999, 21:25-32; Epstein and Butow, CurrentOpinion in Biotechnology 2000, 11:36-41; van Berkum and Holstege, Hedgeet al., Biotechniques 2000, 29:548-560; Current Opinion in Biotechnology2001, 12,:48-52, ). Several methods can be used to isolate the RNA froma population of cells, to prepare cDNAs, to post-amplify the cDNAs ifneeded (low amount of cells), and to label the cDNAs are known (Ausubelet al., Short Protocols in Molecular Biology, Wiley 1999; Wang et al.,Nature Biotechnology 2000, 18:457-459; Mansfield et al., Mol Cell Probes1995, 9:145-56). Several variables need to be controlled for ingene-expression analysis (such as the amount of starting material,enzymatic efficiencies, labeling efficiencies, slide inhomogeneities . .. ) to ensure proper quantitative results. Several calibration methods,using internal (e.g. housekeeping genes like β-actin, cyclophilin,β2-microglobulin, Hypoxanthine phosphoribosyl-transferase I, UbiquitinC, GAPDH, hydroxymethyl-bilane synthase . . . ) or external (“spiking”)controls, are known (Schuchhardt et al., Nucleic Acids Research 1990,28:e47; Tseng et al., Nucleic Acids Research 1991, 29:2549-2557; Yue etal, Nucleic Acids Research 2001, 29:e41). Image analysis, and datacorrection and analysis are integrated into several available softwaretools (Bassett et al., Nature Genetics Supplement 1999, 21:51-55;Quackenbush, Nature Reviews Genetics 2001, 2:418-427).

Preferably, the bringing into presence is realized on a DNA chip whichbears said synthetic DNA fragments according to a specific topography.

The process also applies micro array technology.

The high-density DNA chip allows the evaluating of the expression of agreat number of genes (for example, the whole genes contained in theorganism genome), while the lower-density DNA chip allows the evaluationof the expression of a lower number of genes (often those associatedwith a pathology). These DNA chips are in technological evolution andthey allow the nature and the quantity of some mRNA contained in onecell to be determined in a few hours. The use of these DNA chips hasbeen already considered in a great number of areas in fundamentalresearch but also in applied science, for example, in the follow-up careof cancer patients, people exposed to toxic actions of the environmentand in cases of resistance to triple therapy in patients infected by theAIDS virus. These known DNA chips are thus intended for the analysis ofa determined pathology.

In the process of the invention, the population of synthetic DNAfragments may be composed of oligonucleotides whose size is between 25and 100 b, of products from in vitro enzymatic amplification (PCR), or amixture thereof.

Preferably, the population of synthetic DNA fragments comprises at least50 gene fragments belonging to the family of genes chosen from the groupconstituting the enzymes coding for:

-   -   enzymes with antioxidant functions, enzymes with pro-oxidant        functions, enzymes for the DNA repair, enzymes of the metabolism        of glutathion, stress proteins, proteins implied in apoptosis,        transcription factors, cytokines and chemokines.

Preferably, the population of the synthetic DNA fragments selectedcomprises at least two genes each belonging to one distinct family amongthe precited families.

More preferably, the population of synthetic DNA fragments comprises oneor more fragments of each precited gene families.

As said anti-oxidant enzymes, there may be the following: catalaseXM006202, superoxide dismutase containing manganese X14322, superoxidedismutase containing copper and zinc X81859, thioredoxine-1XM_(—)015718, thioredoxine reductase-1 XM_(—)015673, peroxyredoxine-1XM_(—)011983, metallothioneine-1 X97261, L-ferritine XM_(—)016853,H-ferritine XM_(—)017556, transferrine receptor XM_(—)002788,antioxidant protein 2 NM_(—)004905, ceruloplasmin M13699, lactoferrinM93150, selenoprotein P Z11793, selenoprotein W U67171, frataxin U43747,serum paraoxonase/arylesterase 3 (PON3) L48516.

As enzymes having pro-oxidant functions, there can be, for example, thefollowing: cyclooxygenase-2 M90100, 5-lipooxygenase XM_(—)005818,c-phospholipase A2 XM_(—)007544, phospholipase A alpha D16234,phospholipase D-1 NM_(—)002662, myeloperoxydase XM_(—)008160, nitricoxide synthetase-2 XM_(—)008631, C reactive protein X56692, elastaseM34379, haptoglobin NM_(—)005143, NADH-cytochrome b5 reductase ordiaphorase A1 Y09501.

As said enzymes for the DNA repair, there can be, for example,8-oxoguanine human DNA glycosylase BC000657.

As precited glutathion metabolism enzymes, it can be mentioned forexample, the following: glutathione peroxidase X58295, non-Seglutathione phospholipid hydroperoxide AF090194, gamma-glutamyl cysteinesynthetase NM_(—)001498, and glucose 6-phosphate dehydrogenaseXM_(—)013149, extracellular glutathione peroxidase 2 NM_(—)002083,glutathione peroxidase 4 NM_(—)002085, glutathione reductase X15722,glutathione S-transferase J03746, glutathione synthetase U34683,peroxiredoxin 1 (PRDX1) NM_(—)002574, peroxiredoxin 2 (PRDX2)XM_(—)009063, peroxiredoxin 3 (PRDX3) XM_(—)055573, peroxiredoxin 5(PRDX5) AF197952, thioredoxin 2 (TRX2) AF276920.

As previously cited stress protein, the following may be mentioned, forexample: heat shock protein M11717, heme-oxygenase-1 XM009946, hemeoxygenase-2 D21243, 150 kDa oxygen-regulated protein ORP150 U65785,27-kDa heat shock protein (HSP17) U15590, 40-kDa heat shock protein 1(HSP40) D49547, 110-kDa heat shock protein (HSP110) D89956, ubiquitinM26880.

As said protein implied in apoptosis, one can mention for example, FasLAF287593, CD95 X89101, receptor 1 tumor necrosis factor M32315, Bcl-2NM_(—)000633, GADD153 S40706, GADD45 M60974, RAD50 U63139, RAD51BD13804, RAD52 U12134, RAD54 X97795, p53 AF307851, Fas Ligand 38122.

As said transcription factors, the following may be mentioned: IκB-α,c-Fos V01512; C-Jun J04111, IκB-α M69043, monoamine oxidase A (MAOA)M68840, monoamine oxidase B (MAOB) M69177, peroxisomeproliferative-activated receptor alpha (PPAR-alpha) L02932.

As said cytokines and chemokines, for example, IL-1 BC-008678, IL-6BC-015511, IL-8 AF-385628, IL-1β M15330, IL-2 U25676, and TNF1receptor-associated protein (TRAP1) U12595 can be mentioned.

Said substances are all known and some have received an access number inthe PubMed Central and Gene Bank databases, which is cited above.

It must be noted that this list is not all inclusive, but it doespermit, during the application of several genes coding for several ofthese substances, one single analysis of a group of distinctpathologies, each having an effect that may be important, depending onthe cases Accordingly, this increases the quality of the analysis andmakes it particularly safe, which was not possible until now on thebasis of a single known test. In another embodiment of this invention,the expression of the sets of genes identified by DNA array arequantified using quantitative RT-PCR technologies which are moresuitable when a limited number of genes (usually less than 10) are to bequantified or when starting with fewer materials (e.g. less than 1 mlblood). Total lymphocyte mRNA is purified as above and the genes arequantified using one or a combination of the RT-PCR technologies, asdescribed by S. A. Bustin (Journal of Molecular Endocrinology, 25,169-193, 2000) either in separate reactions or in a multiplex format.The design of the amplimers and the probes, of the PCR conditions, andof the RNA controls, and the different labeling options are described byBustin (ibid.), Graber et al (Current Opinion in Biotechnology 1998,9:14-18), Schweitzer and Kingsmore (Current Opinion in Biotechnology2001, 12:21-27) and Lie and Petropoulos (Current Opinion inBiotechnology 1998, 9:43-48). The data are normalized using internal andexternal controls similarly to what is done with DNA arrays. Results arereported in absolute concentrations (e.g. average number of mRNA perlymphocyte). Range of normal values in the absence of oxidative stresswill be determined as above.

Although described as an independent embodiment, the following subjectmatter should also be understood as preferred embodiment of the abovesubject matter. The present invention relates also to a processcomprising in addition, before said mRNA extraction, an in vitroexposition of the cells of the blood sample to factors generatingoxidative stress, preferably selected from H₂O₂, HOCl, xanthine/xanthineoxidase, glucose/glucose oxidase, phorbol myristate acetate,azo-compounds and thermal stress (+41° C.), which allows the evaluationof the efficiency of the selected synthetic DNA fragments before theirimplementation in a diagnostic test.

The invention also relates to a global evaluation of the SSO by theparallel performance of the single genomic test according to theinvention, of the quantification of blood markers of oxidative stress inthe sample.

The invention also relates to a kit for the detection of oxidativestress for the implementation of the process characterized in that itcomprises:

-   -   at least one DNA chip which bears said population of synthetic        DNA fragments.

Advantageously, this kit comprises moreover, a hybridization solutionadapted to the DNA chip implemented, as well as possibly an adaptedwashing solution. As a control, it may have at least one gene having aconstant expression level in every oxidative stress situation, forexample cyclophilline, GAPDH, some beta-actine and ribosomal RNA or anaverage of several genes. Preferably, this kit presents also a protocolfor every necessary step to realize a hybridization and to proceed tothe detection of obtained hybridization signals, as well as a referencevalue table of expressed levels of DNA fragments deposited on said atleast one DNA chip, these levels corresponding to a positive value ofoxidative stress.

Other methods and forms of realization of the invention are indicated inthe claims.

FIG. 1 shows the distribution curve for antibodies against oxidized LDL.

FIG. 2 shows the parallel determination of oxidative stress markersusing a microarray assay.

A. EXPERIMENTAL CONDITIONS OF OXIDATIVE STRESS AND EXTRACTION OF mRNA

A blood sample (1 ml) taken with heparine as anticoagulant orlymphocytes (10⁷ cells/ml) isolated from blood samples (coming from thekit from Nycorned Lymphoprep, In VitroGen) are contacted at 37° C. byseveral production systems of oxidative stress: 1) hydrogen peroxidewith a concentration ranging from 10⁻⁴ to 10⁻⁶M; ii) xanthine oxidase0,4 U/ml/hypoxanthine 2 mM; iii) hydrochlorure2,2′-azobis(2-amidinipropane) at a concentration of 50 mM. Theincubation period may vary between 30 minutes and 3 hours. At the end ofthe incubation period, the extraction of the RNA messenger is effectedfrom the full blood or from lymphocytes using a commercial kit “μMACSmRNA isolation kit from Miltenyi Biotec Germany”.

B. Development of DNA Chips with Low Density SSO

The DNA chips SSO are created simultaneously using PCR productsgenerated by specific oligonucleotides from genes sequences candidateschosen for example, between those detailed above, as well as frompolynucleotides representing fragments of these genes. The size of thePCR products will be in this example comprised between 400-600 pairs ofbases and the size of polynucleotides will be between 60-80 bases. Thesystem used for the deposition of the products on the glass blade isEuroGridder SDDC-2.

C. Preparation of Fluorescent Probes (Complementary DNA) andHybridization of the DNA Chips SSO

The RNA messengers extracted are transformed into complementary DNA byreverse transcription. The reverse transcriptase is the SuperScript IIsold by Invitrogen/Life Technology. The protocol of the reversetranscription is the one coming with the enzyme. The complementary DNAare then amplified by PCR using the specific oligonucleotides of theselected genes and the polymerase SilverStar (Eurogentec) according tothe protocol given with the enzyme. The labeling is done during theamplification by incorporation of dCTP coupled with either CY3 or eitherCY5 (from Amersham/Pharmacia). The complementary DNA are then puttogether and diluted in a hybridization solution “Dig Easy Hybridizationsolution” sold by Roche Boehringer and deposited on the DNA chips (20 μlper blade, covered by a cover slip of 24×32 mm). The blades are then putindividually in hybridization rooms sold by Corning and incubated during5 to 16 hours at 42° C.

D. Washing of the DNA Chips, Detection and Analysis of the HybridizationSignals

After hybridization, the DNA chips are washed twice for 5 minutessuccessively in solutions 0.2×SSC-0.1% SDS and 0.2×SSC a (1×SSC=0,15 MNaCl−0,015 M citrate sodium, pH: 7,5). The hybridization signals aremeasured with the scanner GenePix 4000A and the results are treated withthe GenePix software 3.0 from Axon Instruments.

E. Validation of the Tests on DNA Chips of Low Density SSO

For each tested gene, reference values are established from a range ofsamples obtained from 50 patients in good health. The quantification ofthe gene expression will be done compared to the basic expression ofgenes like cyclophilline, GAPDH, the beta-actine and the ribosomal RNA.These values are compared to the ones obtained on the blood samplessubmitted to in vitro oxidative stress as described above.

The efficiency of DNA chips SSO is also preferably being tested onpatients having an oxidative stress which has been evidenced and this onthe basis of classical blood dosage.

Once the efficiency of the DNA chips is observed, these can be directlyapplied to diagnose an oxidative stress situation on patients who maypossibly suffer from such a situation.

Material and Methods

A. Detection of Oxidative Stress.

A battery of 22 assays is used to evidence in vivo oxidative stress.They include the determination of antioxidant and trace elements, theanalysis of oxidative damage to lipids and proteins, and theinvestigation of iron metabolism. The normal range for each assay wasestablished on a population of 123 healthy and sedentary individuals(age 21-64 years). Blood samples were drawn on appropriatedanticoagulant and immediately spun. Plasma aliquots were kept at −20° C.until assay.

Antioxidants.

Because it is highly labile, plasma vitamin C or ascorbic acid wasestimated spectrophotometrically using reduction of the dye2,6-dichlorophenolindophenol (Merck, Germany) after stabilization with a10% metaphosphoric acid solution as previously described (Omaye S etal., Methods Enzymol. 62: 3-11; 1979).

Plasma vitamin E or α-tocopherol concentration was assessed byhigh-pressure liquid chromatography (HPLC) on reversed phase column C-18120 (100×4.5 mm i.d.) with an isocratic elution with methanol/water(98:2) and ultraviolet radiation (UV) detection at 280 nm, according toBieri et al. (Am. J. Clin. Nutr. 32: 2143-2149; 1979). As vitamin E iscarried by lipids, vitamin E status was expressed as vitaminE/cholesterol ratio.

Plasma vitamin A concentration was analyzed by HPLC and UV detection at325 nm using kit provided by Chromsystems (Munich, Germany).

Reduced (GSH) and oxidized (GSSG) glutathione were assessed by thecalorimetric determination using the thiol-scavenging reagent,1-methyl-2-vinylpyridinium trifluoromethanesulfonate (BioxytechGSH/GSSG-412, Oxis, USA).

Proteins sulfhydryl (P-SH) groups concentration was assessed in plasmaby the spectrophotometric method using 5,5′-dithio-bis (2-nitrobenzoicacid) (Sigma, Steinheim, Germany) reduction according to Ellman et al.(Arch. Biophys. 82: 70-77; 1959).

Glutathione peroxidase (GPx) and superoxide dismutase (SOD) enzymes wereassessed in the whole blood using a spectrophotometric method adapted toroutine assay (Randox Laboratories, Antrim, UK).

Trace Elements.

Selenium, copper and zinc were measured on plasma by a directgraphite-furnace atomic-absorption-spectroscopic procedure on a SPIRAA-640 (Varian, the Netherlands) equipped with a Zeeman backgroundcorrection (Nève J. et al., in: Bratter P; Schamer P., eds. Traceelements analytical chemistry in medicine and biology. Berlin: Walter deGruyter; 1987: 1-10). The ratio copper/zinc was used as a sensitivemarker of the presence of in vivo oxidative stress (Mezzetti A. et al.,Free Rad. Biol. Med., 25; 676-681; 1998).

Markers of Oxidative Stress.

The concentration of lipid peroxide was determined by reaction of thebiological peroxides with peroxidase and a subsequent color-reactionusing TMB as substrate. After addition of a stop solution, the coloredliquid was measured photometrically at 450 nm.

Oxidized LDL was determined by an Elisa technique developed by Mercodia(Uppsala, Sweden). It was based on the direct sandwich technique inwhich two monoclonal antibodies are directed against separate antigenicdeterminants on the oxidized apolipoprotein B molecule.

Autoantibodies against oxidized LDL (ox-LDL-Ab) were determined with acommercially available enzyme immunoassay (Biomedica Gruppe, Vienna,Austria) initially developed by Tatzber and Esterbauer (in: Rice-Evans,C.; Bellomo, G. eds. Free radicals IX. London: Richelieu Press; 1995:245-262). Copper (Cu²⁺) oxidized LDL was coated onto microtiter stripsas antigen. Autoantibodies, if present in the prediluted plasma, boundspecifically to the antigen. After a washing step, a specific peroxidaseconjugated antihuman IgG antibody detected the presence of boundautoantibodies. After removal of unbound conjugate through washing,tetramethylbenzidine was added to wells as a nontoxic chromogenicsubstrate. The concentration of specific IgG in the sample wasquantitated by an enzyme catalyzed color change detectable on a standardELISA reader.

The degree of protein oxidation was monitored by the method of Levine etal. (Methods Enzymol. 186: 464-478; 1990), which uses the reaction of2,4-dinitrophenylhydrazine (DNPH) with the carbonyl groups of oxidizedproteins. Protein carbonyls were then read at 370 nm and evaluated usinga molar absorption coefficient of 22,000 M⁻¹cm⁻¹.

F. Iron Metabolism

Seric iron was determined after reduction of ferric ions into ferrousions which react with ferrozine reagent to form a red colored complex.Absorbency was read at 572 and 660 nm (kit Merck n° 19725).

The ADVIA Centaur Ferritine measure is a two sites immunodosage(sandwich), using a direct chemiluminescence technology and constantquantity of anti-ferritine antibodies. The first antibody is ananti-ferritine goat polyclonal antibody, coupled with acridinium ester.The second antibody is an anti-ferritine mouse monoclonal antibodycovalently bounded to paramagnetic particles.

Transferrin was analyzed by immunoturbidimetric test using kit fromAptec, catalog n° 53-080040/1.

G. Miscellaneous

Total homocysteine (tHcy) (μmol/L) was measured by HPLC coupled withfluorescence detection as described by Jacob et al. (Ann. Biol. Clin.(Paris), 55: 583-591; 1997).

H. Establishment of Reference Values

When the parameter observed shows a good normal distribution in thepopulation studied, its reference value is defined as the mean±twice thestandard deviation (SD) obtained from the absolute frequency histogramafter defining appropriate classes (Gaussian distribution optimized bysuccessive iterations). Differences between mean concentrations recordedfor different sub-groups (smokers, fruit eaters . . . ) were evaluatedby means of Student's t-test for independent variables. Differences werejudged statistically significant at p <0.05. Correlations werecalculated by multiple regression of the various independent variables.

B. Microarray Experimentation.

The use of DNA arrays (cDNA microarrays or oligonucleotide microarrays)has been proposed a powerful technology to assess antioxidant andprooxidant gene expression. Blood samples obtained at rest from 16healthy adults were collected as control population. With regard to theclassical blood analysis, no significant oxidative stress was detectedin this population (a maximum of 2 abnormal parameters on 6 investigatedassays as shown on table I).

We started an experimental design to analyze the expression profiles oflymphocytes in three in vivo conditions of oxidative stress:

-   -   A. Patient with chronic renal failure; blood samples were        collected before hemodialysis. This test sample was compared to        gene expression profile found in the control population.    -   B. Patient submitted to cardiac surgery associated with        cardio-pulmonary bypass procedure; blood samples were collected        before (reference cDNA in the present situation) and 24 h after        cardiac surgery (test sample).    -   C. Six well-trained athletes (five male and one female)        performed an official half-marathon; blood samples were        collected at rest (reference cDNA in the present situation) and        within 1 hour after the competition (test sample).

The preparation of lymphocytes was started within 1 hour after bloodcollection.

Microarray Preparation

Preparation of the cDNAs: Primary polymerase chain reaction (PCR) wererealized: 10 μl mix of specific primers 1 and 2 (vol. 1:1; cf. tableIIa) and 90 μl PCR mixture containing 20 ng human cDNA pool, 10× EuroTaqDNA polymerase buffer, 2 mM MgCl₂, 0.8 mM dNTP and 0.02 U/μl EuroTaq DNApolymerase (Eurogentec, Belgium). Amplification was then performed withdenaturation for 5 min at 94° C., followed by 40 cycles of denaturationat 94° C. for 1 min, annealing at 55° C. for 30 sec and extension at 72°C. for 1 min.

Amino-modified PCR were realized with 30 μl primary PCR products and 70μl PCR mixture containing 10× EuroTaq DNA polymerase buffer, 2 mM MgCl₂,0.8 mM dNTP and 0.02 U/μl EuroTaq DNA polymerase (Eurogentec, Belgium)added by 20 pmol of reverse primer: 5′-GTC-CGG-GAG-CCA-TG-3′ (seq75) and20 pmol of forward primer: 5′-CGA-CGC-CCG-CTG-ATA-3′ (seq76).Amplification was then performed with denaturation for 5 min at 94° C.,followed by 40 cycles of denaturation at 94° C. for 1 min, annealing at45° C. for 30 sec and extension at 72° C. for 1 min.

Purified amino-modified PCR products and long oligos (˜65 mer; at aconcentration of 10 μM; cf table IIb), were arrayed onto Diaglassmicroscope slides (Advanced Array technology, Namur, Belgium) usingEurogridder SDDC-2 robotics (ESI/Virtek).

A total of 32 cDNAs and 10 long oligos were arrayed in 1 cm² areas(Tables IIa and IIb). Printed arrays were washed once with 0.2% SDS for2 min, twice with distilled water for 2 min, and incubated once for 5min in sodium borohydride solution (NaBH₄ 2.5 mg/ml dissolved in aPBS/EtOH solution (75/25)). The arrays were washed once with distilledwater for 2 min and submerged in distilled water heated to 95° C. for 3min, air dried and then stored in the dark at room temperature.

Preparation of Leukocytes for cDNA Hybridization

EDTA blood was first diluted in PBS Dulbecco's (Gibco,) (1:1). Tenmilliliters of EDTA blood-PBS were carefully layered over 5 ml ofLymphoprep (Axis-Shield, Oslo, Norway) and centrifuge for 20 min at 3000rpm. The erythrocytes were sedimented to the bottom of the tube. Themajority of the leukocytes remained in the plasma layer and wereremoved. The overlay was washed two times with PBS, and the leukocyteswere counted in a Cell-Dyn 1600 (Sequoia-Turner).

Isolation of RNA and cDNA Array Hybridization

Total RNA were isolated from lymphocytes using Rneasy kit (Qiagen,Germany), as described by the manufacturers. Final concentrations weremonitored by spectrophotometry. Reverse transcriptions (RT) wereperformed with 1 μg total RNA, first strand buffer 5×, 0.1 pmol/μl“Specific Primer Mix” (10 μl of all primers 1 and primers 2 added by 290μl Rnase free water), 1.5 mM dNTP (w/o dCTP), 0.025 mM dCTP, 1.25 nMbiotin-labeled and fluorescent-labeled dCTP (PerkinElmer-life sciences,USA), 0.01 M DTT, 1.5 μg oligo dT, 3 μg anchored oligo dT, 1 μlSuperscript II Rnase H-Reverse Transcriptase enzyme (Invitrogen, USA)and 1 μl Rnase OUT (Invitrogen, USA). Biotin and fluorescent-labeledcDNA were pooled, purified and concentrated with microcon-YM30 filter(Millipore, Mass., USA). The labeled probes were then resuspended in 20μl hybridization buffer (5×SSC, 2% SDS, 20% Formamide) added by 0.5 μlDNA salmon sperm and was applied slowly under the cover-slip of the DNAmicroarray. Hybridization was carried out overnight at 42° C. inside ahybridization chamber (Corning, N.Y., USA).

Posthybridization and Cyanine-3 (Cy3™) and Cyanine-5 (Cy5™) TSA

After hybridization, the microarray was washed with 30 ml 0.5×SSC, 0.01%SDS, and then with 30 ml 0.06×SSC, 0.01% SDS. Finally, the microarraywas washed with 0.06×SSC. The signal is amplified by the tyramide signalamplification (TSA) system MICROMAX (Perkin Elmer-life sciences, USA) asdescribed by the manufacturer. Briefly, hybridization signal frombiotin-labeled cDNA was amplified with streptavidin-horseradishperoxidase (HRP) and Cy5-tyramide, while hybridization signal fromfluorescent-labeled cDNA was amplified with anti-fluorescent-HRP andCy3-tyramide. After signal amplification and posthybridization wash, theDNA microarray was air-dried and detected with a laser scanner.

Image Acquisition and Data Analysis

Laser detection of the Cy3 and Cy5 signal on the microarray was acquiredwith a GenePix 4000A (Axon Instruments; CA, USA). The fluorescencesignal intensities and the Cy3/Cy5 ratios were analyzed by the softwareGenePix Pro 3.0 (Axon Instruments; CA, USA).

The background-substracted intensities of all cDNA and oligohybridizations were normalized to each other by the ratios of total Cy3and Cy5-fluorescence values. Three housekeeping genes (cyclophilin,β-actin and GAPDH) have been used as internal controls. Thus,hybridization signals from these control genes in two RNA samples shouldtheoretically be the same. The Cy3-to-Cy5 ratios for each of thesecontrol genes must be closed to 1.

TABLE I OXIDATIVE STRESS LEVEL IN BLOOD SAMPLE OF A CONTROL POPULATIONFROM WHICH LYMPHOCYTES HAVE BEEN ISOLATED FOR DNA MICROARRAY ASSAY. noLDL Lipid Ratio smoker/ Vit.C Ox αLDLox peroxides GSH GSSG GSH/ Sex agesmoker (μg/ml) (U/l) (mlU/ml) (μmol/l) (μmol/l) (μmol/l) GSSG F 22 S12.9 57.61 X 867.8 879.55 2.11 414.99 M 34 NS 10.8 59.64 595.17 356.78602.15 0.25 2406.62 M 27 S 5.58 81.53 119.47 313.55 899.68 5.94 149.4 F35 NS 13.6 49.04 138.27 245.96 817.23 2.75 294.98 F 26 S 9.15 91.27167.42 715.98 803.17 M 47 NS 6.1 107.9 224.76 196.97 845.67 0.18 4610.77M 53 NS 9.18 64.93 135.45 168.33 753 M 34 NS 9.23 61.27 265.19 136.87973.51 0.65 1499.98 F 42 NS 14.3 63.1 195.62 227.54 804.77 1.66 482.48 M51 NS 13.6 64.12 142.03 219.21 906.4 0.16 5824.83 M 39 NS 6.6 56.06354.5 108.74 728.07 1.46 497.56 F 36 NS 21.6 33.93 419.36 169.88 874.120.71 1224.04 M 24 NS 12.3 60.45 491.75 138.61 877.63 0.82 1062.99 F 23NS 1.75 66.8 162.72 629.48 754.27 0.87 866.46 M 39 NS 7.51 73.07 1434.7267.5 982.46 28.15 32.9 M 32 S 8.04 147.1 482.35 72.259 866.45 1.41612.82 Mean 35 10.14 71.11 355.25 302.22 835.51 3.37 1427.20 S.D. 9.64.61 26.38 336.06 232.24 95.84 7.29 1737.16 Normal 4-10 26-117 200-600<400 753-958 1.17-5.32 156-705 range N = 123

TABLE IIA Sequences of human genes selected primers used to detectoxidative stress. GENES ACCESS N^(o) PRIMER 1 PRIMER 2 CatalaseXM_006202 GATATGGATCACATACTTTCAAGCTGG GGTAGGGACAGTTCACAGGTATATG (SEQ IDNO: 1) (SEQ ID NO: 2) Mn superoxide dismutase X14322GTGGTGGTCATATCAATCATAGCATT CATTCCAAATAGCTTTTAGATAATCAG (SEQ ID NO: 3)(SEQ ID NO: 4) Cu/Zn superoxide dismutase X81859 GCAATGTGACTGCTGACAAAGATATGATGCAATGGTCTCCTGAGAG (SEQ ID NO: 5) (SEQ ID NO: 6) Thioredaxin 1XM_015718 TTGTAGTAGTTGACTTCTCAGCC CCACCTTTTGTCCCTTCTTAAAA (SEQ ID NO: 7)(SEQ ID NO: 8) Thioredoxin reductase-1 XM_015673CCTATGACTATGACCTTATCATCATTGG CCTTAATCCTGTGAGGACCAATAAA (SEQ ID NO: 9)(SEQ ID NO: 10) Peroxiredoxin-1 XM_011983 TTCTTTCAGATTTGACCCATCAGATCGGGATTATCTGTTTCACTACCAGGT (SEQ ID NO: 11) (SEQ ID NO: 12) L-ferritinXM_016853 CCAGATTCGTCAGAATTATTCCAC TAAGCTTCACTTCCTCATCTAGG (SEQ ID NO:13) (SEQ ID NO: 14) H-ferritin XM_017556 TCTTCACCAATCTCATGAGGAGAGGTGGTCACATGGTCATCCAATTCTT (SEQ ID NO: 15) (SEQ ID NO: 16) Transferrinreceptor XM_002788 TTTATACACTCCTGTGAATGGATCTATAGCAACATAGTGATCTGGTTCTACAAAG (SEQ ID NO: 17) (SEQ ID NO: 18) Pro-oxidantenzymes: Cyclo-oxygenase-2 M90100 CAGAAATACAACTATCAACAGTTTATCTACGTAGGCAGGAGAACATATAACATTA (SEQ ID NO: 19) (SEQ ID NO: 20) 5-lipoxygenaseXM_005818 TGATATCCAGTTTGATAGTGAAAAAGG GAAGGGAGGAAAATAGGGTTCTC (SEQ IDNO: 21) (SEQ ID NO: 22) Phospholipase A2 XM_007544CTCATCCTGTCATTGGACTACAACC GGTTGTTGCAGACATTGTAATGTG (SEQ ID NO: 23) (SEQID NO: 24) Phospholipase A alpha D16234 CATTAGTGATAAAGATGCCTCTATAGTAGCACATCATAGTAAGCAATAAGTAAGTC (SEQ ID NO: 25) (SEQ ID NO: 26)Phospholipase D1 NM_002662 CTGACATGAGTAATATCATAGAAAATCTGGCCATGTGTTAATTCAATAGTGTAAAGATT (SEQ ID NO: 27) (SEQ ID NO: 28)Myeloperoxidase XM_008160 GTTCCTACAATGACTCAGTGGACCCATACTGCTCCATCAGTTTCCTC (SEQ ID NO: 29) (SEQ ID NO: 30) Nitric oxidesynthase-2A XM-008631 CTTCATGAAGTACATGCAGAATGAATACCCTGTACTTATCCATGCAGACAAC (SEQ ID NO: 31) (SEQ ID NO: 32) Enzymes for theDNA repair: 8-oxoguanine BC000657 CAAGTATGGACACTGACTCAGACTGATGTGCCACATATGGACATCCAC DNA glycosylase (SEQ ID NO: 33) (SEQ ID NO: 34)Enzymes for glutathione metabolism: Glutathione peroxidase X58295GACAAGAGAAGTCGAAGATGGAC TCTTCCTGTAGTGCATTCAGTTC (SEQ ID NO: 35) (SEQ IDNO: 36) Non-Se glutathione AF090194 AGACTCATGGGGCATTCTCTTCTCAGGACCAAAAATAAACACCACAC phospholipid hydroperoxide (SEQ ID NO: 37) (SEQID NO: 38) γ-glutamylcysteine NM_001498 CATTTATAGAAACATTTACTGAGGATGATGTGTCTATTGAGTCATATCGGGATTT synthetase (SEQ ID NO: 39) (SEQ ID NO: 40)Glucose-6-P dehydrogenase XM_013149 TCTATGTGGAGAATGAGAGGTGGGATAAATATAGGGGATGGGCTTGG (SEQ ID NO: 41) (SEQ ID NO: 42) Stress proteins:Heat shock protein-70 M11717 CCATGGTGCTGACCAAGATGAAGGCTGATGTCCTTCTTGTGTTTTC (SEQ ID NO: 42) (SEQ ID NO: 43) Heat shockprotein 70 M15432 CCATGACGAAAGACAACAATCTG AGATGACCTCTTGACACTTGTCC (SEQID NO: 44) (SEQ ID NO: 45) Herne Oxygenase-1 XM_009946CAGGCAGAGGGTGATAGAAGAGG GAGTGTAAGGACCCATCGGAGAA (SEQ ID NO: 46) (SEQ IDNO: 47) Transcription factors: 1κB-α M69043 TCTACACTTAGCCTCTATCCATGGTGAAGGTTTTCTAGTGTCAGCTG (SEQ ID NO: 49) (SEQ ID NO: 50) c-Fos V01512GAGACAGACCAACTAGAAGATGAGA ATAGAAGGACCCAGATAGGTCCA (SEQ ID NO: 51) (SEQID NO: 52) Cytokines: Interleukin-8 AF385628 ATAAAGACATACTCCAAACCTTTCCGGTCCACTCTCAATCACTCTCAG (SEQ ID NO: 53) (SEQ ID NO: 54) Interleukin-6BC015511 TTTAAATATGTGAAGCTGAGTTAATTTATG AATGCCATTTATTGGTATAAAAACC (SEQID NO: 55) (SEQ ID NO: 56) Interleukin-1 BC008678GAGAAGAAAGTAATGACAAAATACCTG AAATTGCATGGTGAAGTCAGTTATA (SEQ ID NO: 57)(SEQ ID NO: 58) House keeping genes: CyclophilinGTCCGGGAGCCATGCCGTGTTCTTCGACATT CGACGCCCGCTGATATGGCCTCCACAATATT (SEQ IDNO: 59) (SEQ ID NO: 60) β-actin CTCTTCCAGCCTTCCTTCCTCACCTTCACCGTTCCAGTTT (SEQ ID NO: 61) (SEQ ID NO: 62) GAPDHCGAGATCCCTCCAAAATCAA TGAGCTTGACAAAGTGGTCG (SEQ ID NO: 63) (SEQ ID NO:64)

TABLE IIb Sequences of human genes selected long oligos used to detectoxidative stress. GENES ACCESS N^(o) LONG OLIGO Anti-oxidant enzymes:Catalase XM_006202GAGCCTGGATGTGGCTCCCGTAGTCAGGGTGGACCTCAGTGAAGTTCTTGACCGCTTTCTTCTGG (SEQID NO: 65) Mn superoxide dismutase X14322GCTGTAACATCTCCCTTTGCCAACGCCTCCTGGTACTTCTCCTCGGTGACGTTCAGGTTGTTCAC (SEQID NO: 66) Thioredoxin 1 XM_015718GGCCCACACCACGTGGCTGAGAAGTCAACTACTACAAGTTTATCACCTGCAGCGTCCAAGGC (SEQ IDNO: 67) Thioredoxin reductase-1 XM_015673CCAGCAAGAAATCCAGCGCACTCCAAAGCGACATAGGATGCTCCAACAACCAGGGTCTTACCCGG (SEQID NO: 68) Peroxiredoxin-1 XM_011983AGCTGTTCCTTCCCAGTAGGGCGCTGGCTTGGAAATCTTCGCTTTGCTTAGGTGCAGGGAGTG (SEQ IDNO: 69) Enzymes for glutathione metabolism: Glutathione peroxidaseX58295 CAAAGTTCCAGCGGATGTCGTGAACCTTCATGGGTTCCCAGAAGAGGCGGTCAGATGTACCCAGG(SEQ ID NO: 70) Non-Se glutathione AF090194GGCTGGGGTGTGTAGCGGAGGTATTTCTTGCCAGATGGGAGCTCTTTGGTGAAGACTCCTTTAGGphospholipid hydroperoxide (SEQ ID NO: 71) γ-glutamylcysteine NM_001498CCTTCCGGCGTTTTCGCATGTTGGCCTCAACTGTATTGAACTCGGACATTGTTCCTCCGTAGGGCsynthetase (SEQ ID NO: 72) Glucose-6-P dehydrogenase XM_013149AGGATGAAGGGCACCCCATCCCACCTCTCATTCTCCACATAGAGGACGACGGCTGCAAAAGTGGC (SEQID NO: 73) Stress proteins: Heme oxygenase-1 XM_009946GGCATAAAGCCCTACAGCAACTGTCGCCACCAGAAAGCTGAGTGTAAGGACCCATCGGAGAAGCG (SEQID NO: 74)Correlation with Sequence Listing

SEQUENCES OF HUMAN GENES SELECTED PRIMERS USED TO DETECT OXIDATIVESTRESS. GENES ACCESS N° PRIMER 1 PRIMER 2 Anti-oxydant enzymes: CatalaseXM_006202 Seq01 Seq02 Mn superoxide dismutase X14322 Seq03 Seq04 Cu/Znsuperoxide dismutase X81859 Seq05 Seq06 Thioredoxin 1 XM_015718 Seq07Seq08 Thioredoxin reductase-1 XM_015673 Seq09 Seq10 Peroxiredoxin-1XM_011983 Seq11 Seq12 L-ferritin XM_016853 Seq13 Seq14 H-ferritinXM_017556 Seq15 Seq16 Transferrin receptor XM_002788 Seq17 Seq18Pro-oxydant enzymes: Cyclo-oxygenase-2 M90100 Seq19 Seq20 5-lipoxygenaseXM_005818 Seq21 Seq22 Phospholipase A2 XM_007544 Seq23 Seq24Phospholipase A alpha D16234 Seq25 Seq26 Phospholipase D1 NM_002662Seq27 Seq28 Myeloperoxidase XM_008160 Seq29 Seq30 Nitric oxidesynthase-2A XM_008631 Seq31 Seq32 Enzymes for the DNA repair:8-oxoguanine DNA BC000657 Seq33 Seq34 glycosylase Enzymes forglutathione metabolism: Glutathione peroxidase X58295 Seq35 Seq36 Non-Seglutathione AF090194 Seq37 Seq38 phospholipid hydroperoxideγ-glutamylcysteine synthetase NM_001498 Seq39 Seq40 Glucose-6-Pdehydrogenase XM_013149 Seq41 Seq42 Stress proteins: Heat shockprotein-70 M11717 Seq43 Seq44 Heat shock protein 70 M15432 Seq45 Seq46Heme oxygenase-1 XM_009946 Seq47 Seq48 Transcription factors: IκB-αM69043 Seq49 Seq50 c-Fos V01512 Seq51 Seq52 Cytokines: Interleukin-8AF385628 Seq53 Seq54 Interleukin-6 BC015511 Seq55 Seq56 Interleukin-1BC008678 Seq57 Seq58 House keeping genes: Cyclophilin Seq59 Seq60β-actin Seq61 Seq62 GAPDH Seq63 Seq64Correlation with Sequence Listing

SEQUENCES OF HUMAN GENES SELECTED LONG OLIGOS USED TO DETECT OXIDATIVESTRESS. GENES ACCESS N° LONG OLIGO Anti-oxydant enzymes: CatalaseXM_006202 Seq65 Mn superoxide dismutase X14322 Seq66 Thioredoxin 1XM_015718 Seq67 Thioredoxin reductase-1 XM_015673 Seq68 Peroxiredoxin-1XM_011983 Seq69 Enzymes for glutathione metabolism: Glutathioneperoxidase X58295 Seq70 Non-Se glutathione phospholipid hydroperoxideAF090194 Seq71 γ-glutamylcysteine synthetase NM_001498 Seq72 Glucose-6-Pdehydrogenase XM_013149 Seq73 Stress proteins: Heme oxygenase-1XM_009946 Seq74

Results

A. Determination of Reference Blood Concentration of Markers Related toOxidative Stress.

All of the antioxidants studied showed a normal distribution. Table IIIlists the reference values for all antioxidants. The mean concentrationfor vitamin C was significantly higher in women than in men (9.32±4.2vs. 6.94±3.75 μg/ml; p<0.0001), but the reverse was true for SOD(710.5±94.5 IU/g Hb in men vs. 666.8±72.5 IU/g Hb in women; p<0,05) anduric acid (334.37±73.78 in men vs. 265.72±89.83 μmol/L in women). Nosignificant difference between the sexes was observed for any of theother parameters (not shown).

FIG. 1 shows a slightly dissymmetric distribution for antibodies againstoxidized LDL. The calculated mean concentration±SD was 424.19±231.26mIU/ml. Men and women showed no significant difference for thisparameter. Values above 650 mIU/ml were recorded in 19.5% of thesubjects (highest value: 1580 mIU/ml). For homocysteine, thedistribution was also slightly dissymmetric in the low values. Themean±SD was 11.71±3.58 μmol/L. Women showed a significantly higherhomocysteine concentration than men (12.92±4.91 vs. 10.60±3.68 μmol/L,p<0.05). Most of our subjects had values near the mean, but 19.5% ofthem had levels between 15 and 30 μmol/L. Only one subject had a levelabove 30 μmol/L.

Regression analysis revealed a weak but significant negative correlationbetween age on the one hand, and vitamins C (r²=0.042; p=0.021) and E(r²=0.063; p=0.005) on the other. It showed a positive correlationbetween age and cholesterol (r²=0.10; p=0.0003). Age did not affect thelevel of homocysteine or of antibodies against oxidized LDL (data notshown). Furthermore, no correlation was observed between either of theserisk factors for cardiovascular disease and any antioxidant level, thecholesterol level, or the LDL-cholesterol level.

Table IV shows the influence of smoking (25% of the subjects smokedbetween 5 and 20 cigarettes a day) on all of the parameters examined inthis study. In smokers, the mean vitamin C level was 31% lower than innon-smokers. The selenium and GPx levels were also significantly reduced(by 9% and 13% respectively), but the effect was less pronounced. Nosignificant difference was detected for the other antioxidants. Insmokers, the, level of antibodies against oxidized LDL was also lowerthan in non-smokers, but the difference was not significant. Smokersadditionally showed a 13.5% higher tHcy level than non-smokers, thisdifference appearing almost significant (p=0.066).

The subjects answered a questionnaire regarding fruit consumption (seedistribution in Table V) and antioxidant intake. Table V shows thatsubjects eating between 1 and 4 fruits a day had a 56.9% higher vitaminC level (significant: p<0.0002) than subjects eating no fruit, and a33.9% higher level than subjects eating 3 to 4 fruits a week(significant: p<0.05). Of the other biochemical parameters, only PSH,selenium, and GPx levels were also higher in subjects eating fruit inlarge amounts. On the other hand, regular fruit consumption correlatedwith a significantly reduced tHcy level. Less than 5% of the subjectsdeclared having taken vitamin complexes in the week prior to bloodsampling. The essential effect was a vitamin C level 29% above the meanshown in Table I.

B. Detection of Oxidative Stress in 4 Conditions.

Parameters described in “material and methods” section were assessed infour categories of people at risk of developing an oxidative stress:patients hospitalized for cardiac surgery with coronary bypass procedure(n=41), patients undergoing 3 times a week a renal dialysis (n=10),athletes regularly racing half-marathon (n=6), and top soccer players(n=13) belonging to the French national team. Blood samples werecollected when all participants were at basal conditions and treatedaccording to procedures assuming the quality as best as possible ofparameters analysis. All data were compared to those obtained in acontrol group of 123 healthy and sedentary volunteers. Underlinednumbers represent abnormal mean values of the studied assay that arelower or higher to the corresponding value observed in the controlgroup. Bold values are within the normal range. However, individualanalysis of data revealed the presence of a large number of abnormalvalues as reflected by the subsequent standard deviation (SD).

Table III shows that the oxidative stress was differently evidenced withrespect to the investigated group.

-   -   In cardiac patients, low values in vitamin C and in SOD were        detected. In contrast, the vitE/cholesterol ratio and GPx levels        were surexpressed. In a less extent, an increase in antibodies        against oxidized LDL and homocysteine was also observed.    -   Dialysis patients are characterized by reduced concentrations in        vitamin C, GPx, SOD, selenium while elevated levels in lipid        peroxides, oxidized LDL and homocysteine were evidenced.    -   People racing half marathons had elevated levels in vitamin C        and in Cu/Zn ratio but low concentrations in VitE/cholesterol        ratio, GPx and seric iron    -   Top soccer players have the worst profile as evidenced by the        large number of abnormal parameters: decrease in vitamin E,        vitE/cholesterol ratio, GSH, GSH/GSSG ratio, zinc and increase        in GPx, GSSG, Cu/Zn ratio, antibodies against oxidized LDL and        oxidized proteins.

TABLE III OXIDATIVE STRESS IN 4 SELECTED POPULATIONS Reference CardiacHalf Top soccer values patients Dialysis marathon players ANTIOXIDANTS n= 123 n = 41 n = 10 n = 6 n = 13 vitamin C (μg/mL)  4-10 5.13 +/− 3.595.46 +/− 2.46 12.46 +/− 2.67  9.72 +/− 3.15 vitamin E (μg/mL)  8-1515.98 +/− 5.44  14.99 +/− 5.67  9.40 +/− 1.90 8.92 +/− 1.74 vitaminE/cholest 4.40-7   8.11 +/− 1.66 7.77 +/− 2.47 5.16 +/− 0.90 4.91 +/−0.83 (mg/g) GPx (lU/g Hb) 30-55 69.03 +/− 17.14  48.7 +/− 18.1534.86 +/− 2.79  50.15 +/− 16.90 SOD (lU/g Hb)  785-1570697.46 +/− 77.53   507.4 +/− 169.83 939.43 +/− 87.24  969.79 +/− 279.69reduced 753-958 ND 814.49 +/− 236.62 835.25 +/− 142.63 717.35 +/− 84.55 glutathione (GSH) (μM) oxidized 1.17-5.32 ND 0.61 +/− 0.86 0.721 +/−0.57  24.96 +/− 50.62 glutathione (GSSG) (μM) ratio GSH/GSSG 156-705 ND4199 +/− 3603 2282 +/− 2575 205.79 +/− 218.49 SH−proteins 216-556 349.85+/− 68.39  300.58 +/− 18.60  ND 474.10 +/− 94.31  (μmol/L) TRACEELEMENTS copper (g/L) 0.70-1.40 ND ND 1.05 +/− 0.34 0.96 +/− 0.12 zinc(g/L) 0.70-1.20 ND ND  0.76 +/− 0.097 0.74 +/− 0.14 ratio Cu/Zn1.00-1.17 ND ND 1.41 +/− 0.54 1.34 +/− 0.35 selenium (μg/100  94-13096.03 +/− 16.73 71.79 +/− 10.88 112.71 +/− 13.97  107.54 +/− 11.92  ml)MARKERS OF OXIDATIVE DAMAGE lipid peroxides  48-306 ND 364.20 +/− 232.46195.14 +/− 231.20 132.95 +/− 55.09   (μmol/L) oxidized LDL (U/L)  26-117ND 159.65 +/− 75.49  39.54 +/− 4.40  48.29 +/− 45.78 antibodies against200-600 415.32 +/− 341.81 148.15 +/− 210.70 210.38 +/− 38.47 610.57 +/− 449.83 ox−LDL (U/L) oxidized proteins 0.224 +/− 0.209 ND0.207 +/− 0.146 ND 0.699 +/− 0.366 (nmol/mg prot) IRON METABOLISM sericiron (μmol/L)  9-33 ND ND 2.28 +/− 0.75 19.89 +/− 6.57  ferritin (ng/mL) 30-300 ND 195.57 +/− 263.93 95.63 +/− 44.59 transferrin (g/L) 1.60-3.50ND 2.53 +/− 0.26 2.33 +/− 0.26 iron capacity of 20-40 ND 37 +/− 17 34+/− 13 saturation of transferrin (%) MISCELLANEOUS Homocysteine 5-1515.36 +/− 3.90  34.01 +/− 17.04 ND 8.91 +/− 2.47 (μmol/L) Underlinednumbers represent abnormal mean values of the studied assay that arelower or higher to the corresponding value observed in the controlgroup. Numbers in bold print are within the normal range. However,individual analysis of data revealed the presence of a large number ofabnormal values reflected by the subsequent standard deviation (SD).

TABLE IV COMPARISON OF OXIDATIVE STRESS BETWEEN SMOKERS AND NO SMOKERS.no smokers (n = 92) Smokers (n = 31) statistics vitamin C (μg/ml)  7.96± 3.83  5.5 ± 3.37 p < 0.005 vitamin A (μg/100 ml)  69.94 ± 18.11  75.95± 21.41 n.s vitamin E (μg/ml) 13.98 ± 3.48 13.74 ± 3.48 n.s Cholesterol(mg/l)  2.01 ± 0.36  2.01 ± 0.33 n.s vitE/cholesterol (mg/g)  7.01 ±1.44  6.86 ± 1.33 n.s selenium (μg/l)  78.56 ± 13.28  72.22 ± 13.63 p <0.05 PSH (μM)  424.91 ± 137.38  413.22 ± 152.62 n.s SOD (Ul/g Hb) 699.08± 90.25 706.71 ± 98.08 n.s GPx (Ul/g Hb)  69.77 ± 21.82  60.83 ± 18.14 p< 0.05 uric acid (mg/l)  54.10 ± 14.60  55.13 ± 13.30 n.s oxidized LDLantibodies  493.55 ± 323.67  393.25 ± 295.33 n.s (mUl/ml) Homocysteine(μM) 11.96 ± 4.21 13.58 ± 5.11 p = 0 . . . 066

TABLE V INFLUENCE OF FRUITS INTAKE ON OXIDATIVE STRESS STATUS. no fruit3-4 fruits/week 1-4 fruits/day (n = 33) (n = 20) (n = 70) vitamin C(μg/ml) 5.48 ± 2.62 6.42 ± 3.31   8.60 ± 4.17*^(,)** vitamin A (μg/100ml) 72.82 ± 22.17 73.25 ± 15.42 70.32 ± 18.68  vitamin E (μg/ml) 14.16 ±3.84  13.06 ± 2.14  14.00 ± 3.48  Cholesterol (mg/l) 2.04 ± 0.38 2.02 ±0.34 2.00 ± 0.34  vitE/cholesterol (mg/g) 6.94 ± 1.25 6.56 ± 1.19 7.04 ±1.53  selenium (μg/l) 73.39 ± 15.21 78.68 ± 14.60 77.35 ± 12.20* PSH(μM) 370.02 ± 77.85  413.22 ± 152.62 444.26 ± 161.08* SOD (Ul/g Hb)717.21 ± 93.11  681.74 ± 81.53  699.07 ± 93.21  GPx (Ul/g Hb) 63.09 ±15.06 74.16 ± 36.92 68.89 ± 16.56* uric acid (mg/l) 57.64 ± 16.36 54.32± 11.49 53.19 ± 13.59  oxidized LDL antibodies 447.53 ± 298.19 499.36 ±440.80 475.64 ± 295.91  (mUl/ml) Homocysteine (μM) 14.75 ± 4.64  10.96 ±3.76* 11.60 ± 4.14** Vitamin C: *p < 0.0002 vs. no fruit; **p < 0.05 vs.3-4 fruits a week. Selenium: *p < 0.05 vs. no fruit; GPx: *p < 0.05 vs.no fruit; PSH: *p < 0.05 vs. no fruit Homocysteine: *p < 0.05 vs. nofruit; **p < 0.005 vs. no fruit Hb = hemoglobin

C. Microarray Experimentation.

Hybridization signals from the housekeeping control genes shouldtheoretically be the same. Experimentations revealed that it is notexactly the case. The ratio varied from 0.3 to 2.1. As proposed by WongK K et al. (Biotechniques, 2001, 30: 670-675), such a problem can beoverwhelmed by using an average ratio of the three housekeeping genesclosed to 1. Thus, the result of internal control for normalizing signalwas similar to that of genes that express at a relatively constant levelin different conditions.

A. Patient with Chronic Renal Failure (Before Hemodialysis).

When compared to the control population, patient regularly submitted torenal dialysis showed an increasing mRNA expression of catalase (CAT),glucose-6-phosphate deshydrogenase (G6PDH), heat shock protein-70 (HSP70) and 5-lipoxygenase (5-LPO) (cf. Table VI).

B. Patient Submitted to Cardiac Surgery Associated with Cardio-pulmonaryBypass Procedure.

When compared to basal condition, a down-regulation of 5 genes wasobserved in lymphocytes isolated 24 h after the surgery. They includesuperoxide dismutase (Mn-SOD), c-phospholipase A2, H-ferritine,Interleukin-8 (IL-8) and nitric oxide synthase 2 (NOS2).

Among these genes, IL-8 was particularly repressed. In contrast, levelof catalase mRNA expression was up-regulated (cf Table VI).

C. Effect of a Half-marathon on Genes Expression.

Physical exercise can elevate core temperature to 44° C. and muscletemperatures up to 45° C. Protection and/or tolerance againstexercise-induced oxidative, heat, cytokine, and inflammatory stress inleukocytes may be in part provided by heat shock proteins (HSPs) family(Fehrenbach E. and Niess A M. Exerc. Immunol. Rev. 5: 57-77; 1999).Using our microarray assay, we have shown a strong upregulation of HSP70(cf. Table VI and FIG. 2). In contrast, a large number of genes aredown-regulated, and more particularly IκB-α.

TABLE VI EXPRESSION OF HUMAN GENES CODING FOR PRO AND ANTIOXIDATIVEPROTEINS MODULATED IN DIFFERENT IN VIVO STRESS SITUATION. GENESHemodialysis Cardiac surgery Half-marathon Catalase A1 5.889 2.054 1.144Peroxyredoxin-1 A2 18.5 1.809 0.304 Thioredoxine reductase-1 A3 11.1441.4  0.356 Mn SOD B1 1.13 0.277 0.88  Glucose-6-P B2 deshydrogenase3.613 1.26  0.746 Thioredoxin-1 B3 1.935 0.926 1.331 Gluthationeperoxidase C1 3.975 0.439 1.669 Non-Se glutathione C2 phospholipidhydroperoxide 5.215 0.825 0.295 γ- glutamylcysteine C3 synthetase 2.9311.055 0.227 Cu/Zn SOD D1 6.546 0.724 0.285 Heme oxygenase-1 D2 4.910.891 0.441 Heat shock protein-70 A2 5.727 1.219 14.227  IκB-α A3 1.9240.551 0.292 Cyclo-oxygenase-2 A4 146.667 0.834 0.342 Phospholipase D-1A6 7.836 0.783 0.499 c-Fos B1 7.885 1.949 1.575 Transferrin receptor B311.367 2.183 0.587 5-lipoxygenase B4 8.613 1.766 0.814 c-phospholipaseA2 B5 1.155 0.247 0.442 Myeloperoxidase B6 1.038 0.51  0.794 L-ferritineC1 1.332 0.789 0.929 H-ferritine C2 0.397 0.21  0.455 IL-8 C3 1.4110.058 0.272 IL-6 C4 0.998 Human 8-oxoguanine DNA C6 glycosylase 0.8390.593 0.336 IL-1 D1 1.839 0.528 0.454 Phospholipase A alpha D2 3.5930.584 0.187 Heat shock protein 70 D3 4.818 1.118 25.662  Nitric oxidesynthase-2 D4 0.737 0.446 0.229 House keeping genes mean 1.336 0.8741.060 The background-substracted intensities of all cDNA and oligohybridizations were normalized to each other by the ratios of total Cy3and Cy5-fluorescence values (only values in fluorescence intensitygreater than 1000 are significant). Three housekeepinggenes(cyclophilin, β-actin and GAPDH) have been used as internalcontrols. The Cy3-to-Cy5 ratios for each of these control genes must beclosed to 1. Underlined numbers represent a down-regulation of the mRNA.Numbers in bold print represent an up-regulation of the mRNA.

1. A method for determining the relative amounts of oxidative stressmarkers in a group of individuals determined to have a risk factor foroxidative stress, which method comprises: (i) measuring the amount of atleast 10 different oxidative stress markers in a sample of whole bloodor component thereof obtained from each individual of the group ofindividuals determined to have a risk factor for oxidative stress; and(ii) comparing the amount of each of the oxidative stress markers in thegroup of individuals determined to have a risk factor for oxidativestress with the amount of each of the oxidative stress markers measuredin a group of healthy individuals; whereupon the relative amounts ofoxidative stress markers in the group of individuals determined to havea risk factor for oxidative stress relative to healthy individuals aredetermined.
 2. A method for the detection of oxidative stress in anindividual having a risk factor for oxidative stress, which methodcomprises: (i) selecting at least two oxidative stress markers, whichhave been determined in accordance with the method of claim 1 toincrease or decrease in individuals with that risk factor relative tohealthy individuals; (ii) measuring the amounts of the at least twooxidative stress markers in a sample of whole blood or component thereofobtained from the individual; and (iii) comparing the amounts of the atleast two oxidative stress markers in the sample obtained from theindividual with the amounts of the same at least two oxidative stressmarkers in healthy individuals; whereupon the individual is determinedto have oxidative stress or not.
 3. The method of claim 2, whichcomprises, in (i), selecting not more than 22 oxidative stress markersthat increase or decrease in individuals with that risk factor relativeto healthy individuals.
 4. The method according to claim 1, wherein therisk factor is selected from: unbalanced diet, smoking habits, exposureto toxic environment, medical surgery, intense physical exercise, anddiseases affecting the kidneys, lungs, heart, skin, brain, joints,gastrointestinal tract, eyes, blood vessels, red blood cells, liver andmultiple organs.
 5. The method according to claim 4, wherein thediseases are selected from transplantation, glomerular nephritis,respiratory distress syndrome, asthma, coronary thrombosis, bums,sunlight exposure, psoriasis, dermatosis, trauma, Parkinson's disease,neurotoxins, dementia, rheumatoid arthritis, diabetes, pancreatitis,endotoxemia, intestinal Ischaemia, cataract, retinopathy, retinaldegeneration, atherosclerosis, Fanconi's anemia, malaria, inflammation,ischaemia-reperfusion, drug toxicity, iron overload, nutritionaldeficiency, alcohol, radiation, cancer, aging, HCV infection and AIDS.6. The method according to claim 1, wherein the oxidative stress markeris selected from the group consisting of: antioxidants, trace elements,indicators of oxidative stress, iron metabolism markers, homocysteine,enzymes having antioxidant functions, enzymes having pro-oxidantfunctions, enzymes for DNA repair, enzymes of the glutathionemetabolism, stress proteins, proteins implied in apoptosis,transcription factors, cytokines and chemokines.
 7. The method accordingto claim 6, wherein the antioxidant is selected from: vitamin A, vitaminC, vitamin E, reduced glutathione (GSH)/oxidized glutathione (GSSG),protein thiols, glutathione peroxidase and superoxide dismutase.
 8. Themethod according to claim 6, wherein the trace element is selected fromselenium, cooper and zinc.
 9. The method of claim 6, wherein theindicator of oxidative stress is selected from8-hydroxy-2′-deoxyguanosine, myeloperoxidase, glucose, glyoxal, and anantibody against oxidized LDL.
 10. The method according to claim 6,wherein the iron metabolism marker is selected from transferrin,ferritin and ceruloplasmin.
 11. The method according to claim 6, whereinthe enzymes having antioxidant function are selected from catalase,Mn-containing superoxide dismutase (SOD), Copper and zinc containingSOD, thioredoxine-1, thioredoxine reductase-1, peroxyredoxine-1,metallothioneine-1, L-ferritine, H-ferritine and transferrine receptor,anti-oxidant protein 2, ceruloplasmin, lactoferrin, selenoprotein P,selenoprotein W, frataxin, serum paraoxonase/arylesterase 1, serumparaoxonase/arylesterase 2, and serum paraoxonase/arylesterase
 3. 12.The method according to claim 6, wherein the enzymes having pro-oxidantfunctions are selected from cyclooxygenase-2,5-lipoxygenase,cphospholipase A2, phospholipase A alpha, phospholipase D-1,myeloperoxidase, nitric oxide synthetase, C reactive protein, elastase,haptoglobin, NADH-cytochrome b5 reductase and diaphorase A1.
 13. Themethod according to claim 6, wherein the enzyme for DNA repair may beselected from is 8-oxoguanine DNA glycosylase.
 14. The method accordingto claim 6, wherein the glutathione metabolism enzyme may is selectedfrom glutathione peroxidase, non-Se glutathione phospholipidhydroperoxide, phospholipid, gamma-glutamyl cysteine synthetase, andglucose 6-phosphate dehydrogenase, extracellular glutathione peroxidase,glutathione peroxidase, glutathione peroxidase 2, glutathione peroxidase4, glutathione reductase, glutathione S-transferase, glutathionesynthetase, peroxiredoxin 1, peroxiredoxin 2, peroxiredoxin 3,peroxiredoxin 5, and thioredoxin
 2. 15. The method according to claim 6,wherein the stress protein is a heat shock protein (HSP) in,heme-oxygenase-1, heme-oxygenase-2, 150 kDa oxygen-regulated protein(ORP)150, 27 kDa HSP27, HSP90A, HSP17, HSP40 or HSP
 110. 16. The methodaccording to claim 6, wherein the protein implied in apoptosis is FasL,CD95, tumor necrosis factor (TNF) receptor 1, Bcl-2, GADD 153, GADD45,RAD50, RAD51B, RAD52, RAD54, p53 or Fas ligand.
 17. The method accordingto claim 6, wherein the transcription factor is selected from NfκB-a,c-Fos, C-jun, IκB-α, monoamine oxidase A, monoamine oxidase B, andperoxisome proliferative-activated receptor α.
 18. The method accordingto claim 6, wherein the cytokine or chemokine is selected from: IL-1,IL-6, IL-8, IL-1beta, IL-2 and TNF1 receptor associated protein.
 19. Themethod according to claim 1, when the risk factor of said individual ishemodialysis, and the oxidative marker is selected from the group ofcatalase, glucose 6 phosphate dehydrogenase, HSP70, 5-lipoxygenase,vitamin C, glutathione peroxidase, SOD, Se, lipid peroxide, oxidized LDLand homocysteine.
 20. The method according to claim 1, when the riskfactor of said individual in cardiac surgery, and the oxidative markeris selected from the group of superoxide dismutase containing manganese,c-phospholipase A2, H-ferritin, IL-8, nitric oxide synthase 2 (NOS2),vitamin C, vitamin E/cholesterol, glutathione peroxidase (GPx),antibodies against LDL and homocysteine.
 21. The method according toclaim 1, when the risk factor of said individual is intense physicalexercise, the oxidative marker is selected from the group of vitamin B,vitamin E/cholesterol, GSH, GSH/GSSG ratio, zinc, GPx, GSSG, copper/zincratio, antibodies against oxidized LDL and oxidized proteins.
 22. Themethod according to claim 1, when the risk factor of said individual isexhaustion due to physical exercise and injuries, the oxidative markeris selected from the group of vitamin E, vitamin E/cholesterol, GSH,GSH/GSSG ratio, zinc, GPx, GSSG; copper/zinc ratio, antibodies againstoxidized LDL and oxidized proteins.
 23. The method according to claim 1,when the risk factor of said individual is smoking, the oxidative markeris selected from the group of vitamin C, Se, GPx, antibodies againstoxidized LDL and homocysteine.
 24. The method according to claim 1,wherein the amount of the oxidative stress marker is determined bymeasuring the concentration of the oxidative marker.
 25. The methodaccording to claim 1, wherein the amount is determined by measuring theconcentration of the gene transcript/mRNA encoding the oxidative marker.26. The method according to claim 25, wherein the amount of at least twooxidative stress markers is determined in parallel.
 27. The methodaccording to claim 26, wherein the amount of oxidative stress marker ismeasured by using a DNA chip.
 28. A process of detecting oxidativestress in a blood sample comprising cells, which method comprises: (i)extracting mRNA from cells from the blood sample, (ii) reversetranscribing the mRNA into cDNA, with labeling of the cDNA, and (iii)contacting the cDNA with a population of synthetic DNA fragments underhybridizing conditions, wherein the population of synthetic DNAfragments hybridizes with the cDNA when present due to gene expressionunder oxidative stress, and simultaneously detecting hybridization,whereupon oxidative stress in a blood sample comprising cells isdetected.
 29. The process according to claim 28, wherein said bloodsample is a preparation of lymphocytes.
 30. The process according toclaim 28, characterized in that step c. is realized on a DNA chip array,which bears said synthetic DNA fragments according to a specifictopography.
 31. The process according to claim 28, characterized in thatthe population of synthetic DNA fragments is composed ofoligonucleotides having a size of between 25 to 100 b.
 32. The processaccording to claim 28, characterized in that the population of thesynthetic DNA fragments is composed of in vitro polymerase chainreaction (PCR) enzymatic amplification products.
 33. The processaccording to claim 28, characterized in that the population of syntheticDNA fragments is composed of oligonucleotides having a size of between25 and 100 b, and of products coming from in vitro PCR enzymaticamplification.
 34. The process according to claim 28, characterized inthat the population of synthetic DNA fragments comprises at least somegene fragments belonging to a family of genes chosen from the groupconstituted by the one coding for: a. enzymes with antioxidantfunctions, enzymes with pro-oxidant functions, b. enzymes for the DNArepair, c. enzymes of the glutathion metabolism, d. stress proteins, e.proteins implied in apoptosis, f. transcription factors, g. cytokines,or h. chemokines.
 35. The process according to claim 34, characterizedin that the population of synthetic DNA fragments comprises at least twogenes, each of which belongs to one of the families of genes a-h. 36.The process according to claim 28, characterized in that it comprises,in addition, before said mRNA extraction, an in vitro exposition of thecells of the blood sample to factors generating oxidative stress. 37.The process according to claim 28, characterized in that it comprises inparallel a quantification of blood markers of oxidative stress.
 38. Themethod according to claim 3, wherein not more than 15 differentoxidative stress markers are selected.
 39. The method according to claim38, wherein not more than 10 different oxidative stress markers areselected.
 40. The method according to claim 39, wherein not more than 5different oxidative stress markers are selected.
 41. The methodaccording to claim 2, wherein the amount of he oxidative stress markeris determined by measuring the concentration of the oxidative marker.